C-type LECTIN transmembrane antigen expressed in human prostate cancer and uses thereof

ABSTRACT

A novel gene (designated PC-LECTIN) that is highly overexpressed in prostate cancer and its encoded protein is described. PC-LECTIN in normal human tissues is restricted to testis, but is highly expressed in prostate cancer. Consequently, PC-LECTIN provides a diagnostic and/or therapeutic target for prostate cancer.

This application is a continuation of U.S. patent application Ser. No.10/897,911, filed Jul. 23, 2004, now U.S. Pat. No. 7,148,337, which is acontinuation of U.S. patent application Ser. No. 10/460,512, filed Jun.11, 2003, now U.S. Pat. No. 7,153,940 which is a divisional of U.S.patent application Ser. No. 09/638,203, filed Aug. 11, 2000, now U.S.Pat. No. 6,602,501, which claims the benefit of U.S. Provisional PatentApplication No. 60/148,935, filed Aug. 12, 1999, the entire contents ofwhich are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention described herein relates to a novel gene and its encodedprotein, termed PC-LECTIN, and to diagnostic and therapeutic methods andcompositions useful in the management of various cancers that expressPC-LECTIN, particularly prostate cancers.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer causes the death of well over ahalf-million people annually, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most common malecancer and is the second leading cause of cancer death in men. In theUnited States alone, well over 40,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, and chemotherapy continue to be the main treatment modalities.Unfortunately, these treatments are ineffective for many and are oftenassociated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the management of this disease. Although the serum PSAassay has been a very useful tool, its specificity and general utilityis widely regarded as lacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic disease progression, includingthe transition from androgen dependence to androgen independence and thedevelopment of metastatic lesions (Klein et al., 1997, Nat. Med. 3:402).More recently identified prostate cancer markers include PCTA-1 (Su etal., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate stem cellantigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95:1735), and STEAP (Hubert et al., 1999, Proc. Natl. Acad. Sci. USA 96:14523).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

SUMMARY OF THE INVENTION

The present invention generally relates to a novel transmembrane antigenoverexpressed in human prostate cancer, designated PC-LECTIN. ThePC-LECTIN antigen is structurally related to hamster layilin (Borowskyand Hynes, J. Cell Biol. 143:429-42, 1998), a member of the C-typelectin proteins. However, PC-LECTIN does not contain the functionaltalin association domain found in layilin, and therefore is likely tohave a different or modified function. The structural features ofPC-LECTIN identify it as a type 1a transmembrane protein with anextracellular N-terminus and intracellular C-terminus. In addition, thePCLECTIN gene product contains an N-terminal signal sequence. Thetransmembrane topology of the PC-LECTIN protein has also beenestablished experimentally.

The distribution of PC-LECTIN gene expression in normal human tissues ishighly restricted to normal testis. In human prostate cancer, thePC-LECTIN gene is highly overexpressed, as no detectable expression ofthis gene occurs in normal prostate. The PC-LECTIN gene thereforeencodes a prostate tumor antigen, which is useful as a diagnostic and/orprognostic marker, and/or may serve as an excellent target for varioustherapeutic approaches such as antibody, vaccine and small moleculetherapies.

Functionally, PC-LECTIN may be involved in invasion, adhesion ormigration. The PC-LECTIN antigen, like Lectin, binds to sugar moieties,opening a further possibility for therapeutic approaches. In oneapproach, carbohydrate molecules may be used to inhibit PC-LECTINbiological activity. The limited expression of PC-LECTIN in the immuneprivileged tissue of the testis (where a blood-testis barrier exists)suggests that negative background effects of immunological and otherPC-LECTIN specific therapeutic strategies (e.g., carbohydrateinhibition) will be minimal. Given the high level expression observed inprostate cancer, it is possible that PC-LECTIN is also expressed inother human cancers, and to that extent, may similarly be useful asdiagnostic and/or prognostic marker of such other cancers, and/or mayserve as a tumor antigen target for the treatment of such other cancers.PC-LECTIN may also be shed into serum following ligand binding oractivation, as has been observed for several known receptors, includingL-Selectin (for review, see: Tedder et al., 1991, Am. J. Respir. Cell.Mol. Biol. 5: 305-306), thereby opening the possibility for serumdetection and related diagnostic methods. Background levels of PC-LECTINwould be expected to be low or absent in view of the blood-testisbarrier and absence of expression in other normal tissues, suggestingthat detection of PC-LECTIN in serum would specifically correlate withthe presence of a tumor.

The invention provides polynucleotides corresponding or complementary toall or part of the PC-LECTIN genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encodingPC-LECTIN proteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, andrelated molecules, polynucleotides or oligonucleotides complementary tothe PC-LECTIN genes or mRNA sequences or parts thereof, andpolynucleotides or oligonucleotides which hybridize to the PC-LECTINgenes, mRNAs, or to PC-LECTIN-encoding polynucleotides. Also providedare means for isolating cDNAs and the genes encoding PC-LECTIN.Recombinant DNA molecules containing PC-LECTIN polynucleotides, cellstransformed or transduced with such molecules, and host vector systemsfor the expression of PC-LECTIN gene products are also provided.

The invention further provides PC-LECTIN proteins and polypeptidefragments thereof, as well as antibodies that bind to PC-LECTIN proteinsand polypeptide fragments thereof. The antibodies of the inventioninclude polyclonal and monoclonal antibodies, murine and other mammalianantibodies, chimeric antibodies, humanized and fully human antibodies,antibodies labeled with a detectable marker, and antibodies conjugatedto radionuclides, toxins or other therapeutic compositions.

The invention further provides methods for detecting the presence ofPC-LECTIN polynucleotides and proteins in various biological samples, aswell as methods for identifying cells that express a PC-LECTIN. Theinvention further provides various therapeutic compositions andstrategies for treating prostate cancer, including particularly,antibody, vaccine and small molecule therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1D. Nucleotide (SEQ ID NO: 1) and deduced amino acid (SEQ ID NO:2) sequences of a full-length cDNA encoding the PC-LECTIN gene. Thestart methionine and putative Kozak sequence are indicated in bold,N-terminal signal sequence is boxed, type-C lectin domains are boxed andshaded, transmembrane domain is underlined.

FIG. 2A. Amino acid sequence alignment of human PC-LECTIN (SEQ ID NO: 2)with the reported sequence of hamster layilin (SEQ ID NO: 3; Borowskyand Hynes, J. Cell Biol:143:42-42, 1998).

FIG. 2B-2C. Nucleotide sequence alignment of human PC-LECTIN cDNA (SEQID NO: 4) with reported cDNA sequence of hamster layilin (SEQ ID NO: 5;Borowsky and Hynes, J. Cell Biol:143:42-42, 1998) (using LALIGN from theBCM Search Launcher).

FIG. 3A. RT-PCR analysis of PC-LECTIN gene expression in prostate cancerxenografts, normal prostate, and other tissues and cell lines, showingexpression in prostate cancer xenografts. Lane 1 is brain; lane 2 isprostate; lane 3 is LAPC-4 AD; lane 4 is LAPC-4 AI; lane 5 is LAPC-9 AD;lane 6 is LAPC-9 AI; lane 7 is HeLa cells; and lane 8 is a negativecontrol.

FIG. 3B. RT-PCR analysis of PC-LECTIN gene expression in varioustissues, showing low level expression in placenta at 30 cycles. Lane 1is brain; lane 2 is heart; lane 3 is kidney; lane 4 is liver; lane 5 islung; lane 6 is pancreas; lane 7 is placenta; and lane 8 is skeletalmuscle.

FIG. 3C. RT-PCR analysis of PC-LECTIN gene expression in normal prostateand other tissues, showing expression in normal testis only at 25 cyclesof amplification, and low level expression in prostate and spleen at 30cycles. Lane 1 is colon; lane 2 is ovary; lane 3 is leukocytes; lane 4is prostate; lane 5 is small intestine; lane 6 is spleen; lane 7 istestis; and lane 8 is thymus.

FIG. 4A. Northern blot analyses of PC-LECTIN expression in variousnormal human tissues, showing no expression of PC-LECTIN in these normaltissues. Lane 1 is heart; lane 2 is brain; lane 3 is placenta; lane 4 islung; lane 5 is liver; lane 6 is skeletal muscle; lane 7 is kidney; andlane 8 is pancreas.

FIG. 4B. Northern blot analyses of PC-LECTIN expression in variousnormal human tissues, showing testis-specific expression of PC-LECTIN innormal tissues. Lane 1 is spleen; lane 2 is thymus; lane 3 is prostate;lane 4 is testis; lane 5 is ovary; lane 6 is small intestine; lane 7 iscolon; and lane 8 is leukocytes.

FIG. 4C. Northern blot analyses of PC-LECTIN expression in prostatecancer xenografts, showing high level expression in all prostate cancerxenografts, with extremely high level expression in the advancedmetastatic prostate tumor xenograft LAPC-9 AD. Lane 1 is LAPC-4 AD; lane2 is LAPC-4 AI; lane 3 is LAPC-9 AD; and lane 4 is LAPC-9 AI.

FIG. 5. Northern blot analysis of PC-LECTIN in prostate cancerxenografts using an SSH fragment probe. The results show that PC-LECTINis highly expressed in tumors that are grown either subcutaneously (sc)or intratibially (it) within the mouse bone. Lanes 1-3 are LAPC-9 AD sc;lanes 4-6 are LAPC-9AD it.

FIG. 6. Northern expression analysis of PC-LECTIN/58P1D12 in tumors of28 day-post castrated males (lane 2) was compared to the expression intumors of intact males (lane 1). Expression is dramatically reduced intumors from castrated males. As a control, expression of a knownandrogen-regulated gene, TMPRSS2, was also shown to be down-regulatedafter castration. These data suggest that PC-LECTIN expression inprostate tumors is dependent on the presence of androgen.

FIG. 7. Cell surface localization of PC-LECTIN antigen. Shown is aphotograph of an exposed western blot of streptavidin-sepharose purifiedcell surface biotinylated 293T cells transfected with vector containingcDNA encoding 6His-tagged PC-LECTIN (lane 2) using an anti-Hismonoclonal antibody. The PC-LECTIN protein was not detected instreptavidin precipitates from non-biotinylated cells transfected withthe same vector (lane 1). Molecular weight markers are indicated inkilodaltons (kD).

FIG. 8A. Western blot showing that anti-HIS antibodies recognizesecreted recombinant PC-LECTIN/58P1D12-AP fusion protein in conditionedmedia. The lanes contain 20 μl conditioned media from unmodified 293Tcells or 293T cells transfected with PC-LECTIN/58P1D12-AP collected 4hours after media change.

FIG. 8B. Western blot showing that anti-alkaline phosphatase antibodiesalso recognize secreted recombinant PC-LECTIN/58P1D12-AP fusion proteinin conditioned media. The lanes contain 20 μl conditioned media fromunmodified 293T cells or 293T cells transfected withPC-LECTIN/58P1D12-AP collected 4 hours after media change, as in FIG.8A.

FIG. 9A. Expression and purification of the extracellular domain ofPC-LECTIN. 293T cells were transfected with a Tag5 secretion expressionvector encoding the extracellular domain of PC-LECTIN with a C-terminal6×His tag. Conditioned medium was subjected to immobilized metalaffinity chromatography using Ni-NTA agarose (Qiagen). The startingconditioned medium, the flow through, and the eluted purified materialwas run on a 10-20% SDS-PAGE gel and silver stained.

FIG. 9B. Conditioned medium from 293T cells transfected as described forFIG. 9A was run on a 10-20% SDS-PAGE gel and transferred tonitrocellulose and subjected to western blotting using an anti-His pAb

FIG. 10A. Immunoprecipitation of PC-LECTIN from Rat1-PC-LECTIN cells.Rat1 cells stably infected with either neo control virus or virusencoding PC-LECTIN were subjected to immunoprecipitation with serum frommice immunized with purified Tag5-PC-LECTIN protein. Western blotanalysis was carried out with an affinity purified rabbit anti-PC-LECTINpeptide pAb.

FIG. 10B. Immunoprecipitation of PC-LECTIN from Rat1-PC-LECTIN cells, asdescribed for FIG. 10A, except that western blot analysis was carriedout with a 1:1000 dilution of immunized mouse serum.

FIG. 11. Expression of PC-LECTIN in recombinant cell lines and testis.Cell lysates of 293T cells transiently transfected with either pCDNA3.1Myc/His PC-LECTIN or empty vector and of Rat1 cells stably infected witheither neo control or PC-LECTIN retrovirus and of normal testis wereseparated by SDS-PAGE and transferred to nitrocellulose for westernanalysis. Indicated with arrows are the 47 kD band representing fulllength PC-LECTIN, the 40 kD extracellular domain, and the 55 kD Myc/Histagged protein.

FIG. 12. Cell surface recognition of PC-LECTIN on Rat1 cells with Tag5PC-LECTIN immunized mouse serum using flow cytometry. Either Rat1-neo(open area) or Rat1-PC-LECTIN cells (5×10⁵; shaded area) were incubatedwith a 1:2000 dilution of Tag5 PC-LECTIN immunized mouse serum. 3,000cells from each sample were analyzed by flow cytometry for cell surfacestaining of PC-LECTIN. Number of events is plotted as a function ofrelative fluorescence.

FIG. 13. Immunohistochemical analysis of PC-LECTIN-transfected 293Tcells labeled with rabbit polyclonal antibody, showing cell surfaceexpression of PC-LECTIN. The antibody did not stain parental 293T cells.

FIG. 14. Expression of PC-LECTIN analyzed using a multi-tissue RNA dotblot (50 samples). The results show significant expression of58P1D12only in testis. Lower expression was also detected in salivary gland,fetal kidney and fetal spleen. The blot also shows extraneous signals inother areas, which are likely to be non-specific since they fall inbetween the rows and columns of the specific signals. Positionsrepresent the following tissues: A1 brain; A2 amygdala; A3 caudatenucleus; A4 cerebellum; A5 cerebral cortex; A6 frontal lobe; A7hippocampus; A8 medulla oblongata; B1 occipital lobe; B2 putamen; B3substantia nigra; B4 temporal lobe; B5 thalamus; B6 sub-thalamicnucleus; B7 spinal cord; C1 heart; C2 aorta; C3 skeletal muscle; C4colon; C5 bladder; C6 uterus; C7 prostate; C8 stomach; D1 testis; D2ovary; D3 pancreas; D4 pituitary gland; D5 adrenal gland; D6 thyroidgland; D7 salivary gland; D8 mammary gland; E1 kidney; E2 liver; E3small intestine; E4 spleen; E5 thymus; E6 peripheral leukocytes; E7lymph node; E8 bone marrow; F1 appendix; F2 lung; F3 trachea; F4placenta; G1 fetal brain; G2 fetal heart; G3 fetal kidney; G4 fetalliver; G5 fetal spleen; G6 fetal thymus; G7 fetal lung.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a novel transmembrane antigen, designatedPC-LECTIN, that is overexpressed in prostate cancer and is a member ofthe C-type lectin family of proteins. Expression in normal adult tissuesis limited to the testis. Expression was found in prostate cancerxenografts, with higher levels in the androgen-dependent prostate cancerxenografts and lower levels in the androgen-independent xenografts. Thisexpression pattern suggests that PC-LECTIN expression in prostate tumorsis dependent on the presence of androgen. PC-LECTIN also shows acarbohydrate binding specificity similar to that observed for the lectinConcanavalin A.

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook etal., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

As used herein, the terms “advanced prostate cancer”, “locally advancedprostate cancer”, “advanced disease” and “locally advanced disease” meanprostate cancers that have extended through the prostate capsule, andare meant to include stage C disease under the American UrologicalAssociation (AUA) system, stage C1-C2 disease under the Whitmore-Jewettsystem, and stage T3-T4 and N+ disease under the TNM (tumor, node,metastasis) system. In general, surgery is not recommended for patientswith locally advanced disease, and these patients have substantiallyless favorable outcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

As used herein, the terms “metastatic prostate cancer” and “metastaticdisease” mean prostate cancers that have spread to regional lymph nodesor to distant sites, and are meant to include stage D disease under theAUA system and stage T×N×M+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is the preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation, and approximately half of thesepatients die within 6 months thereafter. The most common site forprostate cancer metastasis is bone. Prostate cancer bone metastases are,on balance, characteristically osteoblastic rather than osteolytic(i.e., resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

As used herein, the term “polynucleotide” means a polymeric form ofnucleotides of at least 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA.

As used herein, the term “polypeptide” means a polymer of at least 10amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used.

As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” andthe like, used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37° C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55° C., and most preferably to stringenthybridization conditions.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium, citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

In the context of amino acid sequence comparisons, the term “identity”is used to express the percentage of amino acid residues at the samerelative positions that are the same. Also, in this context, the term“homology” is used to express the percentage of amino acid residues atthe same relative positions that are either identical or are similar,using the conserved amino acid criteria os BLAST analysis, as isgenerally understood in the art. For example, % identity values may begenerated by WU-BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996) . Further details regarding amino acid substitutions,which are considered conservative under such criteria, are providedbelow.

Additional definitions are provided throughout the subsections thatfollow.

PC-Lectin Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a PC-LECTIN gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a PC-LECTIN protein and fragments thereof, DNA, RNA, DNA/RNAhybrid, and related molecules, polynucleotides or oligonucleotidescomplementary to a PC-LECTIN gene or mRNA sequence or a part thereof,and polynucleotides or oligonucleotides that hybridize to a PC-LECTINgene, mRNA, or to a PC-LECTIN encoding polynucleotide (collectively,“PC-LECTIN polynucleotides”). As used herein, the PC-LECTIN gene andprotein is meant to include the PC-LECTIN genes and proteinsspecifically described herein and the genes and proteins correspondingto other PC-LECTIN proteins and structurally similar variants of theforegoing. Such other PC-LECTIN proteins and variants will generallyhave coding sequences that are highly homologous to the PC-LECTIN codingsequence, and preferably will share at least about 50% amino acididentity and at least about 60% amino acid homology (using BLASTcriteria), more preferably sharing 70% or greater homology (using BLASTcriteria).

One embodiment of a PC-LECTIN polynucleotide is a PC-LECTINpolynucleotide having the sequence shown in FIG. 1A-D (SEQ ID NO: 1). APC-LECTIN polynucleotide may comprise a polynucleotide having thenucleotide sequence of human PC-LECTIN as shown in FIG. 1A-D (SEQ ID NO:1), wherein T can also be U; a polynucleotide that encodes all or partof the PC-LECTIN protein; a sequence complementary to the foregoing; ora polynucleotide fragment of any of the foregoing. Another embodimentcomprises a polynucleotide having the sequence as shown in FIG. 1A-D(SEQ ID NO: 1), from nucleotide residue number 380 through nucleotideresidue number 1201, from nucleotide residue number 443 throughnucleotide residue number 1018, or from nucleotide residue number 443through nucleotide residue number 1201, wherein T can also be U. Anotherembodiment comprises a polynucleotide encoding a PC-LECTIN polypeptidewhose sequence is encoded by the cDNA contained in the plasmidp58P1D12-2 as deposited with American Type Culture Collection on Mar.10, 1999 as Accession No. 207152. Another embodiment comprises apolynucleotide that is capable of hybridizing under stringenthybridization conditions to the human PC-LECTIN cDNA shown in FIG. 1A-D(SEQ ID NO: 1) or to a polynucleotide fragment thereof.

Typical embodiments of the invention disclosed herein include PC-LECTINpolynucleotides encoding specific portions of the PC-LECTIN mRNAsequence such as those that encode the protein and fragments thereof.For example, representative embodiments of the invention disclosedherein include: polynucleotides encoding about amino acid 1 to aboutamino acid 10 of the PC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO:2), polynucleotides encoding about amino acid 20 to about amino acid 30of the PC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2),polynucleotides encoding about amino acid 30 to about amino acid 40 ofthe PC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2), polynucleotidesencoding about amino acid 40 to about amino acid 50 of the PC-LECTINprotein shown in FIG. 1A-D (SEQ ID NO: 2), polynucleotides encodingabout amino acid 50 to about amino acid 60 of the PC-LECTIN proteinshown in FIG. 1A-D (SEQ ID NO: 2), polynucleotides encoding about aminoacid 60 to about amino acid 70 of the PC-LECTIN protein shown in FIG.1A-D (SEQ ID NO: 2), polynucleotides encoding about amino acid 70 toabout amino acid 80 of the PC-LECTIN protein shown in FIG. 1A-D (SEQ IDNO: 2), polynucleotides encoding about amino acid 80 to about amino acid90 of the PC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2) andpolynucleotides encoding about amino acid 90 to about amino acid 100 ofthe PC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2), etc. Followingthis scheme, polynucleotides (of at least 10 amino acids) encodingportions of the amino acid sequence of amino acids 100-273 of thePC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2) are typicalembodiments of the invention. Polynucleotides encoding larger portionsof the PC-LECTIN protein are also contemplated. For examplepolynucleotides encoding from about amino acid 1 (or 20 or 30 or 40etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the PC-LECTINprotein shown in FIG. 1A-D (SEQ ID NO: 2) may be generated by a varietyof techniques well known in the art.

Additional illustrative embodiments of the invention disclosed hereininclude PC-LECTIN polynucleotide fragments encoding one or more of thebiological motifs contained within the PC-LECTIN protein sequence. Inone embodiment, typical polynucleotide fragments of the invention canencode one or more of the regions of PC-LECTIN that exhibit homology tohamster layilin. In another embodiment of the invention, typicalpolynucleotide fragments can encode one or more of the PC-LECTIN type-Clectin domains or the transmembrane domain, as disclosed in greaterdetail in the text discussing the PC-LECTIN protein and polypeptidesbelow. In yet another embodiment of the invention, typicalpolynucleotide fragments can encode sequences that are unique to one ormore PC-LECTIN alternative splicing variants.

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. As PC-LECTIN is shown to be overexpressed inprostate cancers, these polynucleotides may be used in methods assessingthe status of PC-LECTIN gene products in normal versus canceroustissues. Typically, polynucleotides encoding specific regions of thePC-LECTIN protein may be used to assess the presence of perturbations(such as deletions, insertions, point mutations etc.) in specificregions (such regions containing a transmembrane domain) of thePC-LECTIN gene products. Exemplary assays include both RT-PCR assays aswell as single-strand conformation polymorphism (SSCP) analysis (seee.g. Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both ofwhich utilize polynucleotides encoding specific regions of a protein toexamine these regions within the protein.

Other specifically contemplated embodiments of the invention disclosedherein are genomic DNA, cDNAs, ribozymes, and antisense molecules, aswell as nucleic acid molecules based on an alternative backbone orincluding alternative bases, whether derived from natural sources orsynthesized. For example, antisense molecules can be RNAs or othermolecules, including peptide nucleic acids (PNAs) or non-nucleic acidmolecules such as phosphorothioate derivatives, that specifically bindDNA or RNA in a base pair-dependent manner. A skilled artisan canreadily obtain these classes of nucleic acid molecules using thePC-LECTIN polynucleotides and polynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,PC-LECTIN. See for example, Jack Cohen, OLIGODEOXYNUCLEOTIDES, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The PC-LECTIN antisense oligonucleotides of the presentinvention include derivatives such as S-oligonucleotides(phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra),which exhibit enhanced cancer cell growth inhibitory action. S-oligos(nucleoside phosphorothioates) are isoelectronic analogs of anoligonucleotide (O-oligo) in which a nonbridging oxygen atom of thephosphate group is replaced by a sulfur atom. The S-oligos of thepresent invention may be prepared by treatment of the correspondingO-oligo with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfurtransfer reagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990),the disclosures of which are fully incorporated by reference herein.Additional PC-LECTIN antisense oligonucleotides of the present inventioninclude morpholino antisense oligonucleotides known in the art (see e.g.Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6:169-175).

The PC-LECTIN antisense oligonucleotides of the present inventiontypically may be RNA or DNA that is complementary to and stablyhybridizes with the first 100 N-terminal codons or last 100 C-terminalcodons of the PC-LECTIN genome or the corresponding mRNA. While absolutecomplementarity is not required, high degrees of complementarity arepreferred. Use of an oligonucleotide complementary to this region allowsfor the selective hybridization to PC-LECTIN mRNA and not to mRNAspecifying other regulatory subunits of protein kinase. Preferably, thePC-LECTIN antisense oligonucleotides of the present invention are a 15to 30-mer fragment of the antisense DNA molecule having a sequence thathybridizes to PC-LECTIN mRNA. Optionally, PC-LECTIN antisenseoligonucleotide is a 30-mer oligonucleotide that is complementary to aregion in the first 10 N-terminal codons and last 10 C-terminal codonsof PC-LECTIN. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of PC-LECTIN expression. L. A.Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

Further specific embodiments of this aspect of the invention includeprimers and primer pairs, which allow the specific amplification of thepolynucleotides of the invention or of any specific parts thereof, andprobes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes may be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers can be usedto detect the presence of a PC-LECTIN polynucleotide in a sample and asa means for detecting a cell expressing a PC-LECTIN protein.

Examples of such probes include polypeptides comprising all or part ofthe human PC-LECTIN cDNA sequence shown in FIG. 1A-D (SEQ ID NO: 1).Examples of primer pairs capable of specifically amplifying PC-LECTINmRNAs are also described in the Examples that follow. As will beunderstood by the skilled artisan, a great many different primers andprobes may be prepared based on the sequences provided herein and usedeffectively to amplify and/or detect a PC-LECTIN mRNA.

As used herein, a polynucleotide is said to be “isolated” when it issubstantially separated from contaminant polynucleotides that correspondor are complementary to genes other than the PC-LECTIN gene or thatencode polypeptides other than PC-LECTIN gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated PC-LECTIN polynucleotide.

The PC-LECTIN polynucleotides of the invention are useful for a varietyof purposes, including but not limited to their use as probes andprimers for the amplification and/or detection of the PC-LECTIN gene(s),mRNA(s), or fragments thereof; as reagents for the diagnosis and/orprognosis of prostate cancer and other cancers; as tools for identifyingmolecules that inhibit calcium entry specifically into prostate cells;as coding sequences capable of directing the expression of PC-LECTINpolypeptides; as tools for modulating or inhibiting the expression ofthe PC-LECTIN gene(s) and/or translation of the PC-LECTIN transcript(s);and as therapeutic agents.

Molecular and Biochemical Features of PC-Lectin

As is described further in the Examples that follow, the PC-LECTIN geneand protein have been characterized in a variety of ways. For example,analyses of nucleotide coding and amino acid sequences were conducted inorder to identify conserved structural elements within the PC-LECTINsequence, topological features, and potentially related molecules.RT-PCR and northern blot analyses of PC-LECTIN mRNA expression wereconducted in order to establish the range of normal and canceroustissues expressing the various PC-LECTIN messages. Western blot analysesof PC LECTIN protein expression in experimentally transfected cells wasconducted to determine cell surface localization.

The PC-LECTIN protein is a type 1a transmembrane cell surface protein ofapproximately 252 amino acids (initially expressed as a 273 amino acidsignal sequence-containing precursor protein) with homology to a hamsterprotein termed “layilin”, which in turn shares homology with the C-typelectins (Borowsky and Hynes, J. C II Biol:143:429-42, 1998). PC-LECTINalso shows homology to the lectin domains of galactose-binding proteinthat was initially purified from the hemolymph of Sarcophaga peregrinalarvae following body wall injury, termed Sarcophaga lectin (Komano etal., 980, J. Biol. Chem. 255: 2919-2924), and subsequently cloned(Takahashi et al., 1885, J. Biol. Chem. 22: 12228-12233; Kobayashi etal., 1989, Biochimica et Biophysica Acta 009: 244-250). The cell surfacelocation of the PC-LECTIN protein has been confirmed experimentally, asfurther described in the Examples sections that follow.

The cDNA nucleotide and deduced amino acid sequences of human PC-LECTINare shown in FIG. 1A-D (SEQ ID NO: 1, 2). An alignment of the amino acidsequence of the PC-LECTIN antigen (SEQ ID NO: 2) with the reportedsequence for hamster layilin (SEQ ID NO: 3) is shown in FIG. 2. AlthoughPC-LECTIN bears close homology to hamster layilin (approximately 44.9%identity, over a 265-residue overlap), it diverges significantly in akey functional domain proposed for the layilin protein. Specifically,the PC-LECTIN protein does not have an approximately 10 amino acidsequence found in the layilin structure which represents a domainbelieved to be responsible for the layilin protein's association withthe cytoskeletal protein talin at cell membrane ruffles (Borowsky andHynes, J. Cell Biol:143:429-42, 1998; Critchley et al., Biochem Soc Symp65:79-99, 1999). At the gene level, alignment of the 2550 bp PC LECTINcDNA with the 1747 bp cDNA of hamster layilin cDNA shows homology over a591 bp region. The rest of the PC-LECTIN region is significantlydifferent from layilin, which is reflected in the differences in theamino acid sequence of the c-terminal half of the extracellular domainand the entire cytoplasmic domain. This suggests that while PC LECTINand layilin are related and probably constitute a sub-family of lectins,PC-LECTIN is unlikely to be the human form of layilin.

Layilin's association with talin is hypothesized to function in cellmotility. The absence of the talin association domain in the PC-LECTINstructure suggests that PC-LECTIN may not interact with talin or thecytoskeleton in the same manner as layilin, if at all. In addition tothe absence of the talin association domain, the PC-LECTIN structurecontains inserted and deleted sequence stretches relative to the layilinstructure. The PC-LECTIN expression profile also diverges from thatreported for layilin. Although layilin is reported to be expressed inmultiple mouse tissues (e.g., ovary, lung, spleen, heart, liver,bladder, lymph node, mammary gland, brain, thyroid and kidney) and celllines, PC-LECTIN seems very specific to testis among normal humantissues and is up regulated in prostate cancer. This suggests thatPC-LECTIN could function as a cell adhesion molecule in metastasis andinvasion in prostate cancer and potentially other cancers. Given itsstructural relationship with layilin and other C-type lectins, PC-LECTINis expected to bind to carbohydrate moieties, as has been confirmed.Accordingly, therapeutic strategies utilizing PC-LECTIN-bindingcarbohydrate molecules to interfere with PC-LECTIN activity may betherapeutically useful in the treatment of cancers expressing PC-LECTIN.

PC-LECTIN expression is essentially testis-specific in normal humantissues, as determined by both RT-PCR and northern blot analysis. Incancer, PC-LECTIN mRNA is overexpressed in human prostate tumorxenografts propagated in SCID mice, and in some cases, very high levelexpression is seen. Therefore, given its cell surface localization andits high level expression in prostate cancer, PC-LECTIN has all of thehallmark characteristics of an excellent therapeutic target for thetreatment of prostate cancer. For these same reasons, PC-LECTIN may alsorepresent an ideal diagnostic marker, particularly in relation todiagnostic imaging. Additionally, it is possible that PC-LECTINexpression increases along with progression of the disease and/or inconnection with the emergence of highly aggressive tumors. In thisregard, the LAPC-9 prostate tumor xenograft in which very high levelexpression of PC-LECTIN has been detected was derived from a highlyaggressive osteoblastic bone metastasis of prostate cancer.

Isolation of PC-Lectin-Encoding Nucleic Acid Molecules

The PC-LECTIN cDNA sequences described herein enable the isolation ofother polynucleotides encoding PC-LECTIN gene product(s), as well as theisolation of polynucleotides encoding PC-LECTIN gene product homologues,alternatively spliced isoforms, allelic variants, and mutant forms ofthe PC-LECTIN gene product. Various molecular cloning methods that canbe employed to isolate full length cDNAs encoding a PC-LECTIN gene arewell known (See, for example, Sambrook, J. et al. Molecular Cloning: ALaboratory Manual, 2d edition., Cold Spring Harbor Press, New York,1989; Current Protocols in Molecular Biology. Ausubel et al., Eds.,Wiley and Sons, 995). For example, lambda phage cloning methodologiesmay be conveniently employed, using commercially available cloningsystems (e.g., Lambda ZAP Express, Stratagene). Phage clones containingPC-LECTIN gene cDNAs may be identified by probing with labeled PC-LECTINcDNA or a fragment thereof. For example, in one embodiment, thePC-LECTIN cDNA (FIG. 1A-D; SEQ ID NO: 1) or a portion thereof can besynthesized and used as a probe to retrieve overlapping and full lengthcDNAs corresponding to a PC-LECTIN gene. The PC-LECTIN gene itself maybe isolated by screening genomic DNA libraries, bacterial artificialchromosome libraries (BACs), yeast artificial chromosome libraries(YACs), and the like, with PC-LECTIN DNA probes or primers.

Recombinant DNA Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules con fininga PC-LECTIN polynucleotide, including but not limited to phages,plasmids, phagemids, cosmids, YACs, BACs, as well as various viral andnon-viral vectors well known in the art, and cells transformed ortransfected with such recombinant DNA or RNA molecules. As used herein,a recombinant DNA or RNA molecule is a DNA or RNA molecule that ha beensubjected to molecular manipulation in vitro. Methods for generatingsuch molecules are well known (see, for example, Sambrook et al, 1989,supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a PC-LECTIN polynucleotide within asuitable prokaryotic or eukaryotic host cell. Examples of suitableeukaryotic host cells include a yeast cell, a plant cell, or an animalcell, such as a mammalian cell or an insect cell (e.g., abaculovirus-infectible cell such as an Sf9 cell). Examples of suitablemammalian cells include various prostate cancer cell lines such LnCaP,PC-3, DU145, LAPC-4, TsuPr1, other transfectable or transducibleprostate cancer cell lines, as well as a number of mammalian cellsroutinely used for the expression of recombinant proteins (e.g., COS,CHO, 293, 293T cells). More particularly, a polynucleotide comprisingthe coding sequence of a PC-LECTIN may be used to generate PC-LECTINproteins or fragments thereof using any number of host vector systemsroutinely used and widely known in the art.

A wide range of host vector systems suitable for the expression of PCLECTIN proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, PC LECTIN may be preferably expressed in severalprostate cancer and non-prostate cell lines, including for example 293,293T, rat-1, 3T3, PC-3, LNCaP and TsuPr1. The host vector systems of theinvention are useful for the production of a PC-LECTIN protein orfragment thereof. Such host-vector systems may be employed to study thefunctional properties of PC-LECTIN and PC-LECTIN mutations.

Proteins encoded by the PC-LECTIN genes, or by fragments thereof, willhave a variety of uses, including but not limited to generatingantibodies and in methods for identifying ligands and other agents andcellular constituents that bind to a PC-LECTIN gene product. Antibodiesraised against a PC-LECTIN protein or fragment thereof may be useful indiagnostic and prognostic assays, imaging methodologies (including,particularly, cancer imaging), and therapeutic methods in the managementof human cancers characterized by expression of a PC-LECTIN protein,including but not limited to cancer of the prostate. Variousimmunological assays useful for the detection of PC-LECTIN proteins arecontemplated, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Such antibodies may be labeled and used asimmunological imaging reagents capable of detecting prostate cells(e.g., in radioscintigraphic imaging methods). PC-LECTIN proteins mayalso be particularly useful in generating cancer vaccines, as furtherdescribed below.

PC-Lectin Proteins

Another aspect of the present invention provides PC-LECTIN proteins andpolypeptide fragments thereof. The PC-LECTIN proteins of the inventioninclude those specifically identified herein, as well as allelicvariants, conservative substitution variants and homologs to the extentthat such variants and homologs can be isolated/generated andcharacterized without undue experimentation following the methodsoutlined below. Fusion proteins that combine parts of differentPC-LECTIN proteins or fragments thereof, as well as fusion proteins of aPC-LECTIN protein and a heterologous polypeptide, are also included.Such PC-LECTIN proteins will be collectively referred to as thePC-LECTIN proteins, the proteins of the invention, or PC-LECTIN. As usedherein, the term “PC-LECTIN polypeptide” refers to a polypeptidefragment or a PC-LECTIN protein of at least 10 amino acids, preferablyat least 15 amino acids.

A specific embodiment of a PC-LECTIN protein comprises a polypeptidehaving the amino acid sequence of human PC-LECTIN as shown in FIG. 1A-D(SEQ ID NO: 2), from amino acid residue number 1 through about aminoacid residue number 273 as shown therein. Another specific embodiment ofa PC-LECTIN protein comprises a polypeptide having the amino acidsequence of human PC-LECTIN as shown in FIG. 1A-D (SEQ ID NO: 2), fromabout amino acid residue number 22 through about amino acid residuenumber 273 as shown therein. A specific embodiment of a PC-LECTINfragment comprises a peptide selected from the group comprisingWIGFTYKTA, ATGEHQAFT, FGNCVELQA, NCVELQASA, and DNHGFGNCV (SEQ ID NO:6-10, respectively), or from the group comprising GLWRNGDGQTSGAC (SEQ IDNO: 25), GGPYLYQWNDDRCNM (SEQ ID NO: 26), EARLACESEGGVLL (SEQ ID NO:27), and the extracellular domain of PC-LECTIN (amino acids 22-213 ofSEQ ID NO: 2). Other specific embodiments include one or both of thetype-C lectin domains and/or the transmembrane domain identified in FIG.1A-D (SEQ ID NO: 2).

In general, naturally occurring allelic variants of human PC-LECTIN willshare a high degree of structural identity and homology (e.g., 90% ormore identity). Typically, allelic variants of the PC-LECTIN proteinswill contain conservative amino acid substitutions within the PC-LECTINsequences described herein or will contain a substitution of an aminoacid from a corresponding position in a PC-LECTIN homologue. One classof PC-LECTIN allelic variants will be proteins that share a high degreeof homology with at least a small region of a particular PC-LECTIN aminoacid sequence, but will further contain a radical departure from thesequence, such as a non-conservative substitution, truncation insertionor frame shift.

Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently b interchangeable, as can alanine (A)and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

PC-LECTIN proteins, including variants, comprise at least one epitope incommon with a PC-LECTIN protein having the amino acid sequence of FIG. 1(SEQ ID NO: 2), such that an antibody that specifically binds to aPC-LECTIN protein will also specifically bind to the PC-LECTIN proteinhaving the amino acid sequence of FIG. 1 (SEQ ID NO: 2). One class ofPC-LECTIN protein variants shares 90% or more identity with the aminoacid sequence of FIG. 1 (SEQ ID NO: 2). A more specific class ofPC-LECTIN protein variants comprises a C-type lectin domain. PreferredPC-LECTIN protein variants are capable of binding carbohydrate moieties,particularly with specificity for high mannose residues and/orN-acetylglucosamine.

PC-LECTIN proteins may be embodied in many forms, preferably in isolatedform. As used herein, a protein is said to be “isolated” when physical,mechanical or chemical methods are employed to remove the PC-LECTINprotein from cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated PC-LECTIN protein. A purified PC-LECTINprotein molecule will be substantially free of other proteins ormolecules that impair the binding of PC-LECTIN to antibody or otherligand. The nature and degree of isolation and purification will dependon the intended use. Embodiments of a PC-LECTIN protein include apurified PC-LECTIN protein and a functional, soluble PC-LECTIN protein.In one form, such functional, soluble PC-LECTIN proteins or fragmentsthereof retain the ability to bind antibody or other ligand.

The invention also provides PC-LECTIN polypeptides comprisingbiologically active fragments of the PC-LECTIN amino acid sequence, suchas a polypeptide corresponding to part of the amino acid sequences forPC-LECTIN as shown in FIG. 1A-D (SEQ ID NO: 2). Such polypeptides of theinvention exhibit properties of the PC-LECTIN protein, such as theability to elicit the generation of antibodies that specifically bind anepitope associated with the PC-LECTIN protein.

Embodiments of the invention disclosed herein include a wide variety ofart accepted variants of PC-LECTIN proteins such as polypeptides havingamino acid insertions, deletions and substitutions. PC-LECTIN variantscan be made using methods known in the art such as site-directedmutagenesis, alanine scanning, and PCR mutagenesis. Site-directedmutagenesis [Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller etal., Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells etal., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells etal., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other knowntechniques can be performed on the cloned DNA to produce the PC-LECTINvariant DNA. Scanning amino acid analysis can also be employed toidentify one or more amino acids along a contiguous sequence. Among thepreferred scanning amino acids are relatively small, neutral aminoacids. Such amino acids include alanine, glycine, serine, and cysteine.Alanine is typically a preferred scanning amino acid among this groupbecause it eliminates the side-chain beyond the beta-carbon and is lesslikely to alter the main-chain conformation of the variant. Alanine isalso typically preferred because it is the most common amino acid.Further, it is frequently found in both buried and exposed positions[Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol.Biol., 150:1 (1976)]. If alanine substitution does not yield adequateamounts of variant, an isosteric amino acid can be used.

As discussed above, embodiments of the claimed invention includepolypeptides containing less than the 273 amino acid sequence of thePC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2). For example,representative embodiments of the invention disclosed herein includepolypeptides consisting of about amino acid 1 to about amino acid 10 ofthe PC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2), polypeptidesconsisting of about amino acid 20 to about amino acid 30 of thePC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2), polypeptidesconsisting of about amino acid 30 to about amino acid 40 of thePC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2), polypeptidesconsisting of about amino acid 40 to about amino acid 50 of thePC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2), polypeptidesconsisting of about amino acid 50 to about amino acid 60 of thePC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2), polypeptidesconsisting of about amino acid 60 to about amino acid 70 of thePC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2), polypeptidesconsisting of about amino acid 70 to about amino acid 80 of thePC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2), polypeptidesconsisting of about amino acid 80 to about amino acid 90 of thePC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2) and polypeptidesconsisting of about amino acid 90 to about amino acid 100 of thePC-LECTIN protein shown in FIG. 1A-D (SEQ ID NO: 2), etc. Following thisscheme, polypeptides consisting of portions of the amino acid sequenceof amino acids 100-273 of the PC-LECTIN protein are typical embodimentsof the invention. Polypeptides consisting of larger portions of thePC-LECTIN protein are also contemplated. For example polypeptidesconsisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 30, or 40 or 50 etc.) of the PC-LECTIN protein shown inFIG. 1A-D (SEQ ID NO: 2) may be generated by a variety of techniqueswell known in the art.

Additional illustrative embodiments of the invention disclosed hereininclude PC-LECTIN polypeptides containing the amino acid residues of oneor more of the biological motifs contained within the PC-LECTINpolypeptide sequence as shown in FIG. 1A-D (SEQ ID NO: 2). In oneembodiment, typical polypeptides of the invention can contain one ormore of the regions of PC-LECTIN that exhibit homology to hamsterlayilin, and/or one or more of the transmembrane or C-type lectindomains identified in FIG. 1A-D (SEQ ID NO: 2). In another embodiment,typical polypeptides of the invention can contain one or more of thePC-LECTIN N-glycosylation sites such as NLTK (SEQ ID NO: 33) at residues86-89 (numbering from first amino acid residue shown in FIG. 1), and/orNQST at residues 255-258 (SEQ ID NO: 34). In another embodiment, typicalpolypeptides of the invention can contain one or more of the PC-LECTINcAMP-/cGMP-dependent protein kinase phosphorylation sites such as RKESat residues 266-269 (SEQ ID NO: 35). In another embodiment, typicalpolypeptides of the invention can contain one or more of the PC-LECTINprotein kinase C phosphorylation sites such as SSR at residues 49-51,SEK at residues 141-143, STR at residues 264-266, and/or TRK at residues264-267. In another embodiment, typical polypeptides of the inventioncan contain one or more of the PC-LECTIN casein kinase IIphosphorylation sites such as SFQE at residues 53-56 (SEQ ID NO: 36),SDGD at residues 95-98 (SEQ ID NO: 37), TRKE at residues 265-268 (SEQ IDNO: 38), and/or SGME at residues 269-272 (SEQ ID NO: 39). In anotherembodiment, typical polypeptides of the invention can contain one ormore of the N-myristoylation sites such as GQKVCF at residues 27-32 (SEQID NO: 40), GVLLSL at residues 66-71 (SEQ ID NO: 71), GTGISD at residues91-96 (SEQ ID NO: 42), GISDGD at residues 93-98 (SEQ ID NO: 43), GLWRNGat residues 102-107 (SEQ ID NO: 44), GQTSGA at residues 109-114 (SEQ IDNO: 45), GSEKCV at residues 140-145 (SEQ ID NO: 46), and/or GIIPNL atresidues 212-217 (SEQ ID NO: 47). Related embodiments of theseinventions include polypeptides containing combinations of the differentmotifs discussed above with preferable embodiments being those thatcontain no insertions, deletions or substitutions either within themotifs or the intervening sequences of these polypeptides.

PC-LECTIN polypeptides can be generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the artbased on the amino acid sequences of the human PC-LECTIN proteinsdisclosed herein. Alternatively, recombinant methods can be used togenerate nucleic acid molecules that encode a polypeptide fragment of aPC-LECTIN protein. In this regard, the PC-LECTIN-encoding nucleic acidmolecules described herein provide means for generating definedfragments of PC-LECTIN proteins. PC-LECTIN polypeptides are particularlyuseful in generating and characterizing domain specific antibodies(e.g., antibodies recognizing an extracellular or intracellular epitopeof a PC-LECTIN protein), in identifying agents or cellular factors thatbind to PC-LECTIN or a particular structural domain thereof, and invarious therapeutic contexts, including but not limited to cancervaccines. PC-LECTIN polypeptides containing particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or on the basis of immunogenicity. Fragmentscontaining such structures are particularly useful in generating subunitspecific anti-PC-LECTIN antibodies or in identifying cellular factorsthat bind to PC-LECTIN.

In a specific embodiment described in the examples that follow, asecreted form of PC-LECTIN may be conveniently expressed in 293T cellstransfected with a CMV-driven expression vector encoding PC-LECTIN witha C-terminal 6×His and MYC tag (pcDNA3.1/mycHIS, Invitrogen). Thesecreted HIS-tagged PSCA in the culture media may be purified using anickel column using standard techniques. Alternatively, an AP-tag systemmay be used (see Example 7).

Modifications of PC-LECTIN such as covalent modifications are includedwithin the scope of this invention. One type of covalent modificationincludes reacting targeted amino acid residues of an PC-LECTINpolypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues ofthe PC-LECTIN. Another type of covalent modification of the PC-LECTINpolypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of the polypeptide. “Alteringthe native glycosylation pattern” is intended for purposes herein tomean deleting one or more carbohydrate moieties found in native sequencePC-LECTIN (either by removing the underlying glycosylation site or bydeleting the glycosylation by chemical and/or enzymatic means), and/oradding one or more glycosylation sites that are not present in thenative sequence PC-LECTIN. In addition, the phrase includes qualitativechanges in the glycosylation of the native proteins, involving a changein the nature and proportions of the various carbohydrate moietiespresent. Another type of covalent modification of PC-LECTIN compriseslinking the PC-LECTIN polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

The PC-LECTIN of the present invention may also be modified in a way toform a chimeric molecule comprising PC-LECTIN fused to another,heterologous polypeptide or amino acid sequence. In one embodiment, sucha chimeric molecule comprises a fusion of the PC-LECTIN with apolyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the PC-LECTIN. In analternative embodiment, the chimeric molecule may comprise a fusion ofthe PC-LECTIN with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of an PC-LECTIN polypeptide in place of at least one variableregion within an Ig molecule. In a particularly preferred embodiment,the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

PC-Lectin Antibodies

Another aspect of the invention provides antibodies that bind toPC-LECTIN proteins and polypeptides. The most preferred antibodies willselectively bind to a PC-LECTIN protein and will not bind (or will bindweakly) to non-PC-LECTIN proteins and polypeptides. Anti-PC-LECTINantibodies that are particularly contemplated include monoclonal andpolyclonal antibodies as well as fragments containing theantigen-binding domain and/or one or more complementarity determiningregions of these antibodies. As used herein, an antibody fragment isdefined as at least a portion of the variable region of theimmunoglobulin molecule that binds to its target, i.e., the antigenbinding region.

For some applications, it may be desirable to generate antibodies thatspecifically react with a particular PC-LECTIN protein and/or an epitopewithin a particular structural domain. For example, preferred antibodiesuseful for cancer therapy and diagnostic imaging purposes are thosewhich react with an epitope in an extracellular region of the PC-LECTINprotein as expressed in cancer cells. Such antibodies may be generatedby using the PC-LECTIN proteins described herein, or using peptidesderived from predicted extracellular domains thereof, as an immunogen.In this regard, with reference to the PC-LECTIN protein sequence shownin FIG. 1, regions in the sequence amino-terminal to the transmembranedomain may be selected as used to design appropriate immunogens andscreening reagents for raising and selecting extracellular-specificPC-LECTIN antibodies.

PC-LECTIN antibodies of the invention may be particularly useful inprostate cancer therapeutic strategies, diagnostic and prognosticassays, and imaging methodologies. Similarly, such antibodies may beuseful in the treatment, diagnosis, and/or prognosis of other cancers,to the extent PC-LECTIN is also expressed or overexpressed in othertypes of cancer. The invention provides various immunological assaysuseful for the detection and quantification of PC-LECTIN and mutantPC-LECTIN proteins and polypeptides. Such assays generally comprise oneor more PC-LECTIN antibodies capable of recognizing and binding aPC-LECTIN or mutant PC-LECTIN protein, as appropriate, and may beperformed within various immunological assay formats well known in theart, including but not limited to various types of radioimmunoassays,enzyme-linked immunosorbent assays (ELI SA), enzyme-linkedimmunofluorescent assays (ELIFA), and the like. In addition,immunological imaging methods capable of detecting prostate cancer arealso provided by the invention, including but limited toradioscintigraphic imaging methods using labeled PC-LECTIN antibodies.Such assays may be used clinically in the detection, monitoring, andprognosis of prostate cancer, particularly advanced prostate cancer.

PC-LECTIN antibodies may also be used in methods for purifying PC-LECTINand mutant PC-LECTIN proteins and polypeptides and for isolatingPC-LECTIN homologues and related molecules. For example, in oneembodiment, the method of purifying a PC-LECTIN protein comprisesincubating a PC-LECTIN antibody, which has been coupled to a solidmatrix, with a lysate or other solution containing PC-LECTIN underconditions which permit the PC-LECTIN antibody to bind to PC-LECTIN;washing the solid matrix to eliminate impurities; and eluting thePC-LECTIN from the coupled antibody. Other uses of the PC-LECTINantibodies of the invention include generating anti-idiotypic antibodiesthat mimic the PC-LECTIN protein.

PC-LECTIN antibodies may also be used therapeutically by, for example,modulating or inhibiting the biological activity of a PC-LECTIN proteinor targeting and destroying prostate cancer cells expressing a PC-LECTINprotein. Antibody therapy of prostate and other cancers is morespecifically described in a separate subsection below.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies may be prepared by immunizing a suitablemammalian host using a PC-LECTIN protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). Examples of protein immunogens includerecombinant PC-LECTIN (expressed in a baculovirus system, mammaliansystem, etc.), PC-LECTIN extracellular domain, AP-tagged PC-LECTIN, etc.In addition, fusion proteins of PC-LECTIN may also be used, such as aPC-LECTIN GST-fusion protein. In a particular embodiment, a GST fusionprotein comprising all or most of the open reading frame amino acidsequence of FIG. 1A-D (SEQ ID NO: 2) may be produced and used as animmunogen to generate appropriate antibodies. Cells expressing oroverexpressing PC-LECTIN may also be used for immunizations. Similarly,any cell engineered to express PC-LECTIN may be used. Such strategiesmay result in the production of monoclonal antibodies with enhancedcapacities for recognizing endogenous PC-LECTIN. Another usefulimmunogen comprises PC-LECTIN peptides linked to the plasma membrane ofsheep red blood cells.

The amino acid sequence of PC-LECTIN as shown in FIG. 1A-D (SEQ ID NO:2) may be used to select specific regions of the PC-LECTIN protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of the PC-LECTIN amino acid sequence may be used to identifyhydrophilic regions in the PC-LECTIN structure. Regions of the PC-LECTINprotein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe art, such as Chou-Fasman, Garnier Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Peptides of PC-LECTINpredicted to bind HLA-A2, such as WIGFTYKTA (SEQ ID NO: 6), ATGEHQAFT(SEQ ID NO: 7), FGNCVELQA (SEQ ID NO: 8), NCVELQASA (SEQ ID NO: 9), andDNHGFGNCV (SEQ ID NO: 10), may be selected for the generation ofantibodies. As discussed in the examples below, immunogenicity has beendemonstrated with the peptides GLWRNGDGQTSGAC (SEQ ID NO: 25),GGPYLYQWNDDRCNM (SEQ ID NO: 26), EARLACESEGGVLL (SEQ ID NO: 27), and theextracellular domain of PC-LECTIN (amino acids 22-213 of SEQ ID NO: 2),which were used to generate polyclonal and monoclonal antibodies usingrabbits and mice, respectively.

Methods for preparing a protein or polypeptide for use as an immunogenand for preparing immunogenic conjugates of a protein with a carriersuch as BSA, KLH, or other carrier proteins are well known in the art.In some circumstances, direct conjugation using, for example,carbodiimide reagents may be used; in other instances linking reagentssuch as those supplied by Pierce Chemical Co., Rockford, Ill., may beeffective. Administration of a PC-LECTIN immunogen is conductedgenerally by injection over a suitable period and with use of a suitableadjuvant, as is generally understood in the art. During the immunizationschedule, titers of antibodies can be taken to determine adequacy ofantibody formation.

PC-LECTIN monoclonal antibodies are preferred and may be produced byvarious means well known in the art. For example, immortalized celllines which secrete a desired monoclonal antibody may be prepared usingthe standard hybridoma technology of Kohler and Milstein ormodifications which immortalize producing B cells, a is generally known.The immortalized cell lines secreting the desired antibodies arescreened by immunoassay in which the antigen is the PC-LECTIN protein orPC-LECTIN fragment. When the appropriate immortalized cell culturesecreting the desired antibody is identified, the cells may be expandedand antibodies produced either from in vitro cultures or from ascitesfluid.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the PC-LECTIN protein can also be produced in thecontext of chimeric or CDR grafted antibodies of multiple speciesorigin. Humanized or human PC-LECTIN antibodies may also be produced andare preferred for use in therapeutic contexts. Methods for humanizingmurine and other non-human antibodies by substituting one or more of thenon-human antibody CDRs for corresponding human antibody sequences arewell known (see for example, Jones et al., 1986, Nature 321: 522-525;Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988,Science 239:1534-1536). See also, Carter et al., 1993, Proc. Nat'l Acad.Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296. Methodsfor producing fully human monoclonal antibodies include phage displayand transgenic animal technologies (for review, see Vaughan et al.,1998, Nature Biotechnology 16: 535-539).

Fully human PC-LECTIN monoclonal antibodies may be generated usingcloning technologies employing large human Ig gene combinatoriallibraries (i.e., phage display) (Griffiths and Hoogenboom, Building anin vitro immune system: human antibodies from phage display libraries.In: Protein Engineering of Antibody Molecules for Prophylactic andTherapeutic Applications in Man. Clark, M. (Ed.), Nottingham Academic,pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65-82). Fully human PC-LECTIN monoclonal antibodiesmay also be produced using transgenic mice engineered to contain humanimmunoglobulin gene loci as described in PCT Patent ApplicationWO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997(see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614).This method avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of PC-LECTIN antibodies with a PC-LECTIN protein may beestablished by a number of well known means, including western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,PC-LECTIN proteins, peptides, PC-LECTIN expressing cells or extractsthereof.

A PC-LECTIN antibody or fragment thereof of the invention may be labeledwith a detectable marker or conjugated to a second molecule, such as acytotoxic agent, and used for targeting the second molecule to aPC-LECTIN positive cell (Vitetta, E. S. et al., 1993, Immunotoxintherapy, in DeVita, Jr., V. T. et al., eds., Cancer: Principles andPractice of Oncology, 4th ed., J.B. Lippincott Co., Philadelphia,2624-2636). Examples of cytotoxic agents include, but are not limited toricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethidiumbromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin,Pseudomonas exotoxin (PE) A, PE40, abrin, arbrin A chain, modeccin Achain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin,enomycin, curicin, crotin, calicheamicin, sapaonaria officinalisinhibitor, and glucocorticoid and other chemotherapeutic agents, as wellas radioisotopes such as ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ⁸⁶Re. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. Antibodies may also beconjugated to an anti-cancer pro-drug activating enzyme capable ofconverting the pro-drug to its active form. See, for example, U.S. Pat.No. 4,975,287.

Further, bi-specific antibodies specific for two or more PC-LECTINepitopes may be generated using methods generally known in the art.Further, antibody effector functions may be modified to enhance thetherapeutic effect of PC-LECTIN antibodies on cancer cells. For example,cysteine residues may be engineered into the Fc region, permitting theformation of interchain disulfide bonds and the generation of homodimerswhich may have enhanced capacities for internalization, ADCC and/orcomplement mediated cell killing (see, for example, Caron et al., 1992,J. Exp. Med. 176: 1191-1195; Shopes, 1992, J. Immunol. 148: 2918-2922).Homodimeric antibodies may also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

PC-Lectin Transgenic Animals

Nucleic acids that encode PC-LECTIN or its modified forms can also beused to generate either transgenic animals or “knock out” animals which,in turn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA that is integrated into the genomeof a cell from which a transgenic animal develops. In one embodiment,cDNA encoding PC-LECTIN can be used to clone genomic DNA encodingPC-LECTIN in accordance with established techniques and the genomicsequences used to generate transgenic animals that contain cells thatexpress DNA encoding PC-LECTIN.

Methods for generating transgenic animals, particularly animals such asmice or rats, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009. Typically,particular cells would be targeted for PC-LECTIN transgene incorporationwith tissue-specific enhancers. Transgenic animals that include a copyof a transgene encoding PC-LECTIN introduced into the germ line of theanimal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding PC-LECTIN. Such animals can be usedas tester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PC-LECTIN can be used toconstruct a PC-LECTIN “knock out” animal that has a defective or alteredgene encoding PC-LECTIN as a result of homologous recombination betweenthe endogenous gene encoding PC-LECTIN and altered genomic DNA encodingPC-LECTIN introduced into an embryonic cell of the animal. For example,cDNA encoding PC-LECTIN can be used to clone genomic DNA encodingPC-LECTIN in accordance with established techniques. A portion of thegenomic DNA encoding PC-LECTIN can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration.

Typically, several kilobases of unaltered flanking DNA (both at the 5′and 3′ ends) are included in the vector (see e.g., Thomas and Capecchi,1987, Cell 51:503 for a description of homologous recombinationvectors). The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced DNA hashomologously recombined with the endogenous DNA are selected (see e.g.,Li et al., 1992, Cell 69:915). The selected cells are then injected intoa blastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras (see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed., IRL, Oxford, 1987,pp. 113-152).

A chimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of thePC-LECTIN polypeptide.

Methods for the Detection of PC-Lectin

Another aspect of the present invention relates to methods for detectingPC-LECTIN polynucleotides and PC-LECTIN proteins and variants thereof,as well as methods for identifying a cell that expresses PC-LECTIN.PC-LECTIN appears to be expressed in the LAPC xenografts that arederived from lymph-node and bone metastasis of prostate cancer, and theexpression profile of PC-LECTIN makes it a potential diagnostic markerfor metastasized disease. In this context, the status of PC-LECTIN geneproducts may provide information useful for predicting a variety offactors including susceptibility to advanced stage disease, rate ofprogression, and/or tumor aggressiveness. As discussed in detail below,the status of PC-LECTIN gene products in patient samples may be analyzedby a variety protocols that are well known in the art includingimmunohistochemical analysis, the variety of northern blottingtechniques including in situ hybridization, RT-PCR analysis (for exampleon laser capture micro-dissected samples), western blot analysis andtissue array analysis.

More particularly, the invention provides assays for the detection ofPC-LECTIN polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable PC-LECTIN polynucleotides include, for example, aPC-LECTIN gene or fragments thereof, PC-LECTIN mRNA, alternative splicevariant PC-LECTIN mRNAs, and recombinant DNA or RNA molecules containinga PC-LECTIN polynucleotide. A number of methods for amplifying and/ordetecting the presence of PC-LECTIN polynucleotides are well known inthe art and may be employed in the practice of this aspect of theinvention.

In one embodiment, a method for detecting a PC-LECTIN mRNA in abiological sample comprises producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing PC-LECTIN polynucleotides as sense and antisense primers toamplify PC-LECTIN cDNAs therein; and detecting the presence of theamplified PC-LECTIN cDNA. Optionally, the sequence of the amplifiedPC-LECTIN cDNA can be determined. In another embodiment, a method ofdetecting a PC-LECTIN gene in a biological sample comprises firstisolating genomic DNA from the sample; amplifying the isolated genomicDNA using PC-LECTIN polynucleotides as sense and antisense primers toamplify the PC-LECTIN gene therein; and detecting the presence of theamplified PC-LECTIN gene. Any number of appropriate sense and antisenseprobe combinations may be designed from the nucleotide sequencesprovided for the PC-LECTIN (FIG. 1A-D; SEQ ID NO: 1) and used for thispurpose.

The invention also provides assays for detecting the presence of aPC-LECTIN protein in a tissue of other biological sample such as serum,bone, prostate, and other tissues, urine, cell preparations, and thelike. Methods for detecting a PC-LECTIN protein are also well known andinclude, for example, immunoprecipitation, immunohistochemical analysis,western blot analysis, molecular binding assays, ELISA, ELIFA and thelike. For example, in one embodiment, a method of detecting the presenceof a PC-LECTIN protein in a biological sample comprises first contactingthe sample with a PC-LECTIN antibody, a PC-LECTIN-reactive fragmentthereof, or a recombinant protein containing an antigen binding regionof a PC-LECTIN antibody; and then detecting the binding of PC-LECTINprotein in the sample thereto.

Methods for identifying a cell that expresses PC-LECTIN are alsoprovided. In one embodiment, an assay for identifying a cell thatexpresses a PC-LECTIN gene comprises detecting the presence of PC-LECTINmRNA in the cell. Methods for the detection of particular mRNAs in cellsare well known and include, for example, hybridization assays usingcomplementary DNA probes (such as in situ hybridization using labeledPC-LECTIN riboprobes, northern blot and related techniques) and variousnucleic acid amplification assays (such as RT-PCR using complementaryprimers specific for PC-LECTIN, and other amplification type detectionmethods, such as, for example, branched DNA, SISBA, TMA and the like).Alternatively, an assay for identifying a cell that expresses aPC-LECTIN gene comprises detecting the presence of PC-LECTIN protein inthe cell or secreted by the cell. Various methods for the detection ofproteins are well known in the art and may be employed for the detectionof PC-LECTIN proteins and PC-LECTIN expressing cells.

PC-LECTIN expression analysis may also be useful as a tool foridentifying and evaluating agents that modulate PC-LECTIN geneexpression. For example, PC-LECTIN expression is restricted to normaltestis, as well as to prostate cancer, and PC-LECTIN may also beexpressed in other cancers. Identification of a molecule or biologicalagent that could inhibit PC-LECTIN expression or over-expression incancer cells may be of therapeutic value. Such an agent may beidentified by using a screen that quantifies PC-LECTIN expression byRT-PCR, nucleic acid hybridization or antibody binding.

Monitoring the Status of Pc-Lectin and its Products

Assays that evaluate the status of the PC-LECTIN gene and PC-LECTIN geneproducts in an individual may provide information on the growth oroncogenic potential of a biological sample from this individual. Forexample, because PC-LECTIN mRNA is so highly expressed in prostatecancers, and not in most normal tissue, assays that evaluate therelative levels of PC-LECTIN mRNA transcripts or proteins in abiological sample may be used to diagnose a disease associated withPC-LECTIN dysregulation, such as cancer, and may provide prognosticinformation useful in defining appropriate therapeutic options.Similarly, assays that evaluate the integrity PC-LECTIN nucleotide andamino acid sequences in a biological sample, may also be used in thiscontext.

The finding that PC-LECTIN mRNA is so highly expressed in prostatecancers, and not in most normal tissue, provides evidence that this geneis associated with dysregulated cell growth and therefore identifiesthis gene and its products as targets that the skilled artisan can useto evaluate biological samples from individuals suspected of having adisease associated with PC-LECTIN dysregulation. In another example,because the expression of PC-LECTIN is normally restricted to testis,one can also evaluate biological samples taken from other tissues todetect PC-LECTIN expression as an indication of metastasis. In thiscontext, the evaluation of the expression status of PC-LECTIN gene andits products can be used to gain information on the disease potential ofa tissue sample. The terms “expression status” in this context is usedto broadly refer to the variety of factors involved in the expression,function and regulation of a gene and its products such as the level ofmRNA expression, the integrity of the expressed gene products (such asthe nucleic and amino acid sequences) and transcriptional andtranslational modifications to these molecules.

The expression status of PC-LECTIN may provide information useful forpredicting susceptibility to particular disease stages, progression,and/or tumor aggressiveness. The invention provides methods and assaysfor determining PC-LECTIN expression status and diagnosing cancers thatexpress PC-LECTIN, such as cancers of the prostate, breast, bladder,lung, bone, colon, pancreatic, testicular, cervical and ovarian cancers.PC-LECTIN expression status in patient samples may be analyzed by anumber of means well known in the art, including without limitation,immunohistochemical analysis, in situ hybridization, RT-PCR analysis onlaser capture micro-dissected samples, western blot analysis of clinicalsamples and cell lines, and tissue array analysis. Typical protocols forevaluating the expression status of the PC-LECTIN gene and gene productscan be found, for example in Current Protocols In Molecular Biology,Units 2 [Northern Blotting], 4 [Southern Blotting], [Immunoblotting] and18 [PCR Analysis], Frederick M. Ausubul et al. eds., 1995.

In one aspect, the invention provides methods for monitoring PC-LECTINgene products by determining the status of PC-LECTIN gene productsexpressed by cells in a test tissue sample from an individual suspectedof having a disease associated with dysregulated cell growth (such ashyperplasia or cancer) and then comparing the status so determined tothe status of PC-LECTIN gene products in a corresponding normal sample,the presence of aberrant PC-LECTIN gene products in the test samplerelative to the normal sample providing an indication of the presence ofdysregulated cell growth within the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in PC-LECTIN mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of PC-LECTIN mRNA may, for example,be evaluated in tissue samples including but not limited to colon, lung,prostate, pancreas, bladder, breast, ovary, cervix, testis, head andneck, brain, stomach, bone, etc. The presence of significant PC-LECTINexpression in any of these tissues may be useful to indicate theemergence, presence and/or severity of these cancers or a metastasis ofcancer originating in another tissue, since the corresponding normaltissues do not express PC-LECTIN mRNA or express it at lower levels.

In a related embodiment, PC-LECTIN expression status may be determinedat the protein level rather than at the nucleic acid level. For example,such a method or assay would comprise determining the level of PC-LECTINprotein expressed by cells in a test tissue sample and comparing thelevel so determined to the level of PC-LECTIN expressed in acorresponding normal sample. In one embodiment, the presence ofPC-LECTIN protein is evaluated, for example, using immunohistochemicalmethods. PC-LECTIN antibodies or binding partners capable of detectingPC-LECTIN protein expression may be used in a variety of assay formatswell known in the art for this purpose.

In other related embodiments, one can evaluate the integrity PC-LECTINnucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like. Such embodiments areuseful because perturbations in the nucleotide and amino acid sequencesare observed in a large number of proteins associated with a growthdysregulated phenotype (see e.g. Marrogi et al., J. Cutan. Pathol.26(8): 369-378 (1999)). In this context, a wide variety of assays forobserving perturbations in nucleotide and amino acid sequences are wellknown in the art. For example, the size and structure of nucleic acid oramino acid sequences of PC-LECTIN gene products may be observed by thenorthern, Southern, western, PCR and DNA sequencing protocols discussedherein. In addition, other methods for observing perturbations innucleotide and amino acid sequences such as single strand conformationpolymorphism analysis are well known in the art (see e.g. U.S. Pat. Nos.5,382,510 and 5,952,170).

In another embodiment, one can examine the methylation status of thePC-LECTIN gene in a biological sample. Aberrant demethylation and/orhypermethylation of CpG islands in gene 5′ regulatory regions frequentlyoccurs in immortalized and transformed cells and can result in alteredexpression of various genes. For example, promoter hypermethylation ofthe pi-class glutathione S-transferase (a protein expressed in normalprostate but not expressed in >90% of prostate carcinomas) appears topermanently silence transcription of this gene and is the mostfrequently detected genomic alteration in prostate carcinomas (De Marzoet al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, thisalteration is present in at least 70% of cases of high-grade prostaticintraepithelial neoplasia (PIN) (Brooks et al, Cancer Epidemiol.Biomarkers Prev., 1998, 7:531-536).

In another example, expression of the LAGE-I tumor specific gene (whichis not expressed in normal prostate but is expressed in 25-50% ofprostate cancers) is induced by deoxy-azacytidine in lymphoblastoidcells, suggesting that tumoral expression is due to demethylation (Letheet al., 1998, Int. J. Cancer 76(6): 903-908). In this context, a varietyof assays for examining methylation status of a gene are well known inthe art. For example, one can utilize in Southern hybridizationapproaches methylation-sensitive restriction enzymes which can notcleave sequences that contain methylated CpG sites in order to assessthe overall methylation status of CpG islands.

In addition, MSP (methylation specific PCR) can rapidly profile themethylation status of all the CpG sites present in a CpG island of agiven gene. This procedure involves initial modification of DNA bysodium bisulfite (which will convert all unmethylated cytosines touracil) followed by amplification using primers specific for methylatedversus unmethylated DNA. Protocols involving methylation interferencecan also be found for example in Current Protocols In Molecular Biology,Units 12, Frederick M. Ausubel et al. eds., 1995.

In another related embodiment, the invention provides assays useful indetermining the presence of cancer in an individual, comprisingdetecting a significant change in the PC-LECTIN alternative splicevariants expressed in a test cell or tissue sample relative toexpression levels in the corresponding normal cell or tissue. Themonitoring of alternative splice variants of PC-LECTIN is useful becausechanges in the alternative splicing of proteins is suggested as one ofthe steps in a series of events that lead to the progression of cancers(see e.g. Carstens et al., Oncogene 15(250: 3059-3065 (1997)).

Gene amplification provides an additional method of assessing the statusof PC-LECTIN. Gene amplification may be measured in a sample directly,for example, by conventional Southern blotting, northern blotting toquantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci.USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

In addition to the tissues discussed above, peripheral blood may beconveniently assayed for the presence of cancer cells, including but notlimited to prostate cancers, using RT-PCR to detect PC-LECTINexpression. The presence of RT-PCR amplifiable PC-LECTIN mRNA providesan indication of the presence of the cancer. RT-PCR detection assays fortumor cells in peripheral blood are currently being evaluated for use inthe diagnosis and management of a number of human solid tumors. In theprostate cancer field, these include RT-PCR assays for the detection ofcells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13: 1195-2000; Heston etal., 1995, Clin. Chem. 41: 1687-1688). RT-PCR assays are well known inthe art.

A related aspect of the invention is directed to predictingsusceptibility to developing cancer in an individual. In one embodiment,a method for predicting susceptibility to cancer comprises detectingPC-LECTIN mRNA or PC-LECTIN protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of PC-LECTINmRNA expression present is proportional to the degree of susceptibility.In a specific embodiment, the presence of PC-LECTIN in prostate tissueis examined, with the presence of PC-LECTIN in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). In a closely related embodiment, one canevaluate the integrity PC-LECTIN nucleotide and amino acid sequences ina biological sample in order to identify perturbations in the structureof these molecules such as insertions, deletions, substitutions and thelike, with the presence of one or more perturbations in PC-LECTIN geneproducts in the sample providing an indication of cancer susceptibility(or the emergence or existence of a tumor).

Yet another related aspect of the invention is directed to methods forgauging tumor aggressiveness. In one embodiment, a method for gaugingaggressiveness of a tumor comprises determining the level of PC-LECTINmRNA or PC-LECTIN protein expressed by cells in a sample of the tumor,comparing the level so determined to the level of PC-LECTIN mRNA orPC-LECTIN protein expressed in a corresponding normal tissue taken fromthe same individual or a normal tissue reference sample, wherein thedegree of PC-LECTIN mRNA or PC-LECTIN protein expression in the tumorsample relative to the normal sample indicates the degree ofaggressiveness. In a specific embodiment, aggressiveness of prostatetumors is evaluated by determining the extent to which PC-LECTIN isexpressed in the tumor cells, with higher expression levels indicatingmore aggressive tumors. In a closely related embodiment, one canevaluate the integrity PC-LECTIN nucleotide and amino acid sequences ina biological sample in order to identify perturbations in the structureof these molecules such as insertions, deletions, substitutions and thelike, with the presence of one or more perturbations indicating moreaggressive tumors.

Yet another related aspect of the invention is directed to methods forobserving the progression of a malignancy in an individual over time. Inone embodiment, methods for observing the progression of a malignancy inan individual over time comprise determining the level of PC-LECTIN mRNAor PC-LECTIN protein expressed by cells in a sample of the tumor,comparing the level so determined to the level of PC-LECTIN mRNA orPC-LECTIN protein expressed in an equivalent tissue sample taken fromthe same individual at a different time, wherein the degree of PC-LECTINmRNA or PC-LECTIN protein expression in the tumor sample over timeprovides information on the progression of the cancer. In a specificembodiment, the progression of a cancer is evaluated by determining theextent to which PC-LECTIN expression in the tumor cells alters overtime, with higher expression levels indicating a progression of thecancer. In a closely related embodiment, one can evaluate the integrityPC-LECTIN nucleotide and amino acid sequences in a biological sample inorder to identify perturbations in the structure of these molecules suchas insertions, deletions, substitutions and the like, with the presenceof one or more perturbations indicating a progression of the cancer.

The above diagnostic approaches may be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention disclosed herein isdirected to methods for observing a coincidence between the expressionof PC-LECTIN gene and PC-LECTIN gene products (or perturbations inPC-LECTIN gene and PC-LECTIN gene products) and a factor that isassociated with malignancy as a means of diagnosing and prognosticatingthe status of a tissue sample. In this context, a wide variety offactors associated with malignancy may be utilized such as theexpression of genes otherwise associated with malignancy (including PSA,PSCA and PSM expression) as well as gross cytological observations (seee.g. Bocking et al., 1984, Anal. Quant. Cytol. 6(2):74-88; Eptsein,1995, Hum. Pathol. 1995 February; 26(2): 223-9; Thorson et al., 1998,Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol.23(8):918-24). Methods for observing a coincidence between theexpression of PC-LECTIN gene and PC-LECTIN gene products (orperturbations in PC-LECTIN gene and PC-LECTIN gene products) and anadditional factor that is associated with malignancy are useful, forexample, because the presence of a set or constellation of specificfactors that coincide provides information crucial for diagnosing andprognosticating the status of a tissue sample.

In a typical embodiment, methods for observing a coincidence between theexpression of PC-LECTIN gene and PC-LECTIN gene products (orperturbations in PC-LECTIN gene and PC-LECTIN gene products) and afactor that is associated with malignancy entails detecting theoverexpression of PC-LECTIN mRNA or protein in a tissue sample,detecting the overexpression of PSA mRNA or protein in a tissue sample,and observing a coincidence of PC-LECTIN mRNA or protein and PSA mRNA orprotein overexpression. In a specific embodiment, the expression ofPC-LECTIN and PSA mRNA in prostate tissue is examined. In a preferredembodiment, the coincidence of PC-LECTIN and PSA mRNA overexpression inthe sample provides an indication of prostate cancer, prostate cancersusceptibility or the emergence or existence of a prostate tumor.

Methods for detecting and quantifying the expression of PC-LECTIN mRNAor protein are described herein and use standard nucleic acid andprotein detection and quantification technologies well known in the art.Standard methods for the detection and quantification of PC-LECTIN mRNAinclude in situ hybridization using labeled PC-LECTIN riboprobes,northern blot and related techniques using PC-LECTIN polynucleotideprobes, RT-PCR analysis using primers specific for PC-LECTIN, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like. In a specific embodiment,semi-quantitative RT-PCR may be used to detect and quantify PC-LECTINmRNA expression as described in the Examples that follow. Any number ofprimers capable of amplifying PC-LECTIN may be used for this purpose,including but not limited to the various primer sets specificallydescribed herein. Standard methods for the detection and quantificationof protein may be used for this purpose. In a specific embodiment,polyclonal or monoclonal antibodies specifically reactive with thewild-type PC-LECTIN protein may be used in an immunohistochemical assayof biopsied tissue.

Identifying Molecules that Interact with PC-Lectin

The PC-LECTIN protein sequences disclosed herein allow the skilledartisan to identify molecules that interact with them via any one of avariety of art accepted protocols. For example one can utilize one ofthe variety of so-called interaction trap systems (also referred to asthe “two-hybrid assay”). In such systems, molecules that interactreconstitute a transcription factor and direct expression of a reportergene, the expression of which is then assayed. Typical systems identifyprotein-protein interactions in vivo through reconstitution of aeukaryotic transcriptional activator and are disclosed for example inU.S. Pat. Nos. 5,955,280, 5,925,523, 5,846,722 and 6,004,746.

Alternatively one can identify molecules that interact with PC-LECTINprotein sequences by screening peptide libraries. In such methods,peptides that bind to selected receptor molecules such as PC-LECTIN areidentified by screening libraries that encode a random or controlledcollection of amino acids. Peptides encoded by the libraries areexpressed as fusion proteins of bacteriophage coat proteins, andbacteriophage particles are then screened against the receptors ofinterest. Peptides having a wide variety of uses, such as therapeutic ordiagnostic reagents, may thus be identified without any priorinformation on the structure of the expected ligand or receptormolecule. Typical peptide libraries and screening methods that can beused to identify molecules that interact with PC-LECTIN proteinsequences are disclosed for example in U.S. Pat. Nos. 5,723,286 and5,733,731.

Alternatively, cell lines expressing PC-LECTIN can be used to identifyprotein-protein interactions mediated by PC-LECTIN. This possibility canbe examined using immunoprecipitation techniques as shown by others(Hamilton B J, et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51).Typically PC-LECTIN protein can be immunoprecipitated from PC-LECTINexpressing prostate cancer cell lines using anti-PC-LECTIN antibodies.Alternatively, antibodies against His-tag can be used in a cell lineengineered to express PC-LECTIN (vectors mentioned above). Theimmunoprecipitated complex can be examined for protein association byprocedures such as western blotting, ³⁵S-methionine labeling ofproteins, protein microsequencing, silver staining and two dimensionalgel electrophoresis.

Related embodiments of such screening assays include methods foridentifying small molecules that interact with PC-LECTIN. For example,small molecules can be identified that interfere with lectin binding tocarbohydrate moieties on other molecules, such as glycoproteins. Typicalmethods are discussed for example in U.S. Pat. No. 5,928,868 and includemethods for forming hybrid ligands in which at least one ligand is asmall molecule. In an illustrative embodiment, the hybrid ligand isintroduced into cells that in turn contain a first and a secondexpression vector. Each expression vector includes DNA for expressing ahybrid protein that encodes a target protein linked to a coding sequencefor a transcriptional module. The cells further contains a reportergene, the expression of which is conditioned on the proximity of thefirst and second hybrid proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown hybrid protein is identified.

A typical embodiment of this invention consists of a method of screeningfor a molecule that interacts with a PC-LECTIN amino acid sequence shownin FIG. 1A-D (SEQ ID NO: 2), comprising the steps of contacting apopulation of molecules with the PC-LECTIN amino acid sequence, allowingthe population of molecules and the PC-LECTIN amino acid sequence tointeract under conditions that facilitate an interaction, determiningthe presence of a molecule that interacts with the PC-LECTIN amino acidsequence and then separating molecules that do not interact with thePC-LECTIN amino acid sequence from molecules that do interact with thePC-LECTIN amino acid sequence. In a specific embodiment, the methodfurther includes purifying a molecule that interacts with the PC-LECTINamino acid sequence. In a preferred embodiment, the PC-LECTIN amino acidsequence is contacted with a library of peptides.

Therapeutic Methods and Compositions

The identification of PC-LECTIN as a prostate cancer protein, opens anumber of therapeutic approaches to the treatment of prostate cancers.As discussed above, PC-LECTIN binds sugar moieties and may be involvedin invasion, adhesion or migration. In addition, PC-LECTIN presentsepitopes at the cell surface that can be targeted for therapy.

The expression profile of PC-LECTIN is reminiscent of the MAGEs, PSA andPMSA, which are tissue-specific genes that are up-regulated in melanomasand other cancers (Van den Eynde and Boon, Int J Clin Lab Res. 27:81-86,1997). Due to their tissue-specific expression and high expressionlevels in cancer, these molecules are currently being investigated astargets for cancer vaccines (Durrant, Anticancer Drugs 8:727-733, 1997;Reynolds et al., Int J Cancer 72:972-976, 1997). The expression patternof PC-LECTIN provides evidence that it is likewise an ideal target for acancer vaccine approach to prostate cancer, as its expression is notdetected in most normal tissues. Its structural features as a potentialcalcium transporter also provides evidence that PC-LECTIN may be a smallmolecule target, as well as a target for antibody-based therapeuticstrategies. The therapeutic strategy can be designed to inhibit thecalcium transporter function of the molecule or to target the PC-LECTINmolecule itself.

Accordingly, therapeutic approaches targeting extracellular portions ofPC-LECTIN, or aimed at inhibiting the activity of the PC-LECTIN protein,are expected to be useful for patients suffering from prostate cancerand other cancers expressing PC-LECTIN. The therapeutic approaches aimedat inhibiting the activity of the PC-LECTIN protein generally fall intotwo classes. One class comprises various methods for inhibiting thebinding or association of the PC-LECTIN protein with its binding partneror with other proteins. Another class comprises a variety of methods forinhibiting the transcription of the PC-LECTIN gene or translation ofPC-LECTIN mRNA.

PC-LECTIN as a Cell Surface Target for Antibody-Based Therapy

The structural features of PC-LECTIN indicate that this molecule islikely a cell surface antigen, providing an attractive target forantibody-based therapeutic strategies. Because PC-LECTIN is expressed oncancer cells and not on most normal cells, systemic administration ofPC-LECTIN-immunoreactive compositions would be expected to exhibitexcellent sensitivity without toxic, non-specific and/or non-targeteffects caused by binding of the immunotherapeutic molecule tonon-target organs and tissues. Antibodies specifically reactive withextracellular domains of PC-LECTIN can be useful to treatPC-LECTIN-expressing cancers systemically, either as conjugates with atoxin or therapeutic agent, or as naked antibodies capable of inhibitingcell proliferation or function.

PC-LECTIN antibodies can be introduced into a patient such that theantibody binds to PC-LECTIN on the cancer cells and mediates thedestruction of the cells and the tumor and/or inhibits the growth of thecells or the tumor. Mechanisms by which such antibodies exert atherapeutic effect may include complement-mediated cytolysis,antibody-dependent cellular cytotoxicity, modulating the physiologicalfunction of PC-LECTIN, inhibiting ligand binding or signal transductionpathways, modulating tumor cell differentiation, altering tumorangiogenesis factor profiles, and/or by inducing apoptosis. PC-LECTINantibodies can be conjugated to toxic or therapeutic agents and used todeliver the toxic or therapeutic agent directly to PC-LECTIN-bearingtumor cells. Examples of toxic agents include, but are not limited to,calchemicin, maytansinoids, radioisotopes such as ¹³¹I, ytrium, andbismuth.

Cancer immunotherapy using anti-PC-LECTIN antibodies may follow theteachings generated from various approaches that have been successfullyemployed in the treatment of other types of cancer, including but notlimited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol.18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186;Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk etal., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al.,1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhonget al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al.,1994, Cancer Res. 54:6160-6166); Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin, such as the conjugation of 131I to anti-CD20antibodies (e.g., Rituxan™, IDEC Pharmaceuticals Corp.), while othersinvolve co-administration of antibodies and other therapeutic agents,such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Fortreatment of prostate cancer, for example, PC-LECTIN antibodies can beadministered in conjunction with radiation, chemotherapy or hormoneablation.

Although PC-LECTIN antibody therapy may be useful for all stages ofcancer, antibody therapy may be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionmay be indicated for patients who have received previously one or morechemotherapy, while combining the antibody therapy of the invention witha chemotherapeutic or radiation regimen may be preferred for patientswho have not received chemotherapeutic treatment. Additionally, antibodytherapy may enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

It may be desirable for some cancer patients to be evaluated for thepresence and level of PC-LECTIN expression, preferably usingimmunohistochemical assessments of tumor tissue, quantitative PC-LECTINimaging, or other techniques capable of reliably indicating the presenceand degree of PC-LECTIN expression. Immunohistochemical analysis oftumor biopsies or surgical specimens may be preferred for this purpose.Methods for immunohistochemical analysis of tumor tissues are well knownin the art.

Anti-PC-LECTIN monoclonal antibodies useful in treating prostate andother cancers include those that are capable of initiating a potentimmune response against the tumor and those that are capable of directcytotoxicity. In this regard, anti-PC-LECTIN monoclonal antibodies(mAbs) may elicit tumor cell lysis by either complement-mediated orantibody-dependent cell cytotoxicity (ADCC) mechanisms, both of whichrequire an intact Fc portion of the immunoglobulin molecule forinteraction with effector cell Fc receptor sites or complement proteins.In addition, anti-PC-LECTIN mAbs that exert a direct biological effecton tumor growth are useful in the practice of the invention. Potentialmechanisms by which such directly cytotoxic mAbs may act includeinhibition of cell growth, modulation of cellular differentiation,modulation of tumor angiogenesis factor profiles, and the induction ofapoptosis. The mechanism by which a particular anti-PC-LECTIN mAb exertsan anti-tumor effect may be evaluated using any number of in vitroassays designed to determine ADCC, ADMMC, complement-mediated celllysis, and so forth, as is generally known in the art.

The use of murine or other non-human monoclonal antibodies, orhuman/mouse chimeric mAbs may induce moderate to strong immune responsesin some patients. In some cases, this will result in clearance of theantibody from circulation and reduced efficacy. In the most severecases, such an immune response may lead to the extensive formation ofimmune complexes which, potentially, can cause renal failure.Accordingly, preferred monoclonal antibodies used in the practice of thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target PC-LECTINantigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-PC-LECTIN mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails may have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination mayexhibit synergistic therapeutic effects. In addition, the administrationof anti-PC-LECTIN mAbs may be combined with other therapeutic agents,including but not limited to various chemotherapeutic agents,androgen-blockers, and immune modulators (e.g., IL-2, GM-CSF). Theanti-PC-LECTIN mAbs may be administered in their “naked” or unconjugatedform, or may have therapeutic agents conjugated to them.

The anti-PC-LECTIN antibody formulations may be administered via anyroute capable of delivering the antibodies to the tumor site.Potentially effective routes of administration include, but are notlimited to, intravenous, intraperitoneal, intramuscular, intratumor,intradermal, and the like. Treatment will generally involve the repeatedadministration of the anti-PC-LECTIN antibody preparation via anacceptable route of administration such as intravenous injection (IV),typically at a dose in the range of about 0.1 to about 10 mg/kg bodyweight. Doses in the range of 10-500 mg mAb per week may be effectiveand well tolerated.

Based on clinical experience with the Herceptin mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV followed by weekly doses of about 2 mg/kgIV of the anti-PC-LECTIN mAb preparation may represent an acceptabledosing regimen. Preferably, the initial loading dose is administered asa 90 minute or longer infusion. The periodic maintenance dose may beadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. However, as one of skill in the art willunderstand, various factors will influence the ideal dose regimen in aparticular case. Such factors may include, for example, the bindingaffinity and half life of the Ab or mAbs used, the degree of PC-LECTINexpression in the patient, the extent of circulating shed PC-LECTINantigen, the desired steady-state antibody concentration level,frequency of treatment, and the influence of chemotherapeutic agentsused in combination with the treatment method of the invention.

Optimally, patients should be evaluated for the level of circulatingshed PC-LECTIN antigen in serum in order to assist in the determinationof the most effective dosing regimen and related factors. Suchevaluations may also be used for monitoring purposes throughout therapy,and may be useful to gauge therapeutic success in combination withevaluating other parameters (such as serum PSA levels in prostate cancertherapy).

Inhibition of PC-LECTIN Protein Function

The invention includes various methods and compositions for inhibitingthe binding of PC-LECTIN to its binding partner or ligand, or itsassociation with other protein(s) as well as methods for inhibitingPC-LECTIN function.

Inhibition of PC-LECTIN With Intracellular Antibodies

In one approach, recombinant vectors encoding single chain antibodiesthat specifically bind to PC-LECTIN may be introduced into PC-LECTINexpressing cells via gene transfer technologies, wherein the encodedsingle chain anti-PC-LECTIN antibody is expressed intracellularly, bindsto PC-LECTIN protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, may bespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatmentwill be focused. This technology has been successfully applied in theart (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13).Intrabodies have been shown to virtually eliminate the expression ofotherwise abundant cell surface receptors. See, for example, Richardsonet al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al.,1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther.1: 332-337.

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies may beexpressed as a single chain variable region fragment joined to the lightchain constant region. Well known intracellular trafficking signals maybe engineered into recombinant polynucleotide vectors encoding suchsingle chain antibodies in order to precisely target the expressedintrabody to the desired intracellular compartment. For example,intrabodies targeted to the endoplasmic reticulum (ER) may be engineeredto incorporate a leader peptide and, optionally, a C-terminal ERretention signal, such as the KDEL amino acid motif. Intrabodiesintended to exert activity in the nucleus may be engineered to include anuclear localization signal. Lipid moieties may be joined to intrabodiesin order to tether the intrabody to the cytosolic side of the plasmamembrane. Intrabodies may also be targeted to exert function in thecytosol. For example, cytosolic intrabodies may be used to sequesterfactors within the cytosol, thereby preventing them from beingtransported to their natural cellular destination.

In one embodiment, PC-LECTIN intrabodies are designed to bindspecifically to a particular PC-LECTIN domain. For example, cytosolicintrabodies that specifically bind to the PC-LECTIN protein may be usedto prevent PC-LECTIN from gaining access to the nucleus, therebypreventing it from exerting any biological activity within the nucleus(e.g., preventing PC-LECTIN from forming transcription complexes withother factors).

In order to specifically direct the expression of such intrabodies toparticular tumor cells, the transcription of the intrabody may be placedunder the regulatory control of an appropriate tumor-specific promoterand/or enhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer may beutilized (See, for example, U.S. Pat. No. 5,919,652).

Inhibition of PC-LECTIN With Recombinant Proteins

In another approach, recombinant molecules that are capable of bindingto PC-LECTIN thereby preventing PC-LECTIN from accessing/binding to itsbinding partner(s) or associating with other protein(s) are used toinhibit PC-LECTIN function. Such recombinant molecules may, for example,contain the reactive part(s) of a PC-LECTIN specific antibody molecule.In a particular embodiment, the PC-LECTIN binding domain of a PC-LECTINbinding partner may be engineered into a dimeric fusion proteincomprising two PC-LECTIN ligand binding domains linked to the Fc portionof a human IgG, such as human IgG1. Such IgG portion may contain, forexample, the C_(H)2 and C_(H)3 domains and the hinge region, but not theC_(H)1 domain. Such dimeric fusion proteins may be administered insoluble form to patients suffering from a cancer associated with theexpression of PC-LECTIN, including but not limited to prostate, breast,bladder, lung, bone, colon, pancreatic, testicular, cervical and ovariancancers, where the dimeric fusion protein specifically binds toPC-LECTIN thereby blocking PC-LECTIN interaction with a binding partner.Such dimeric fusion proteins may be further combined into multimericproteins using known antibody linking technologies.

Inhibition of PC-LECTIN Transcription or Translation

Within another class of therapeutic approaches, the invention providesvarious methods and compositions for inhibiting the transcription of thePC-LECTIN gene. Similarly, the invention also provides methods andcompositions for inhibiting the translation of PC-LECTIN mRNA intoprotein.

In one approach, a method of inhibiting the transcription of thePC-LECTIN gene comprises contacting the PC-LECTIN gene with a PC-LECTINantisense polynucleotide. In another approach, a method of inhibitingPC-LECTIN mRNA translation comprises contacting the PC-LECTIN mRNA withan antisense polynucleotide. In another approach, a PC-LECTIN specificribozyme may be used to cleave the PC-LECTIN message, thereby inhibitingtranslation. Such antisense and ribozyme based methods may also bedirected to the regulatory regions of the PC-LECTIN gene, such as thePC-LECTIN promoter and/or enhancer elements. Similarly, proteins capableof inhibiting a PC-LECTIN gene transcription factor may be used toinhibit PC-LECTIN mRNA transcription. The various polynucleotides andcompositions useful in the aforementioned methods have been describedabove. The use of antisense and ribozyme molecules to inhibittranscription and translation is well known in the art.

Other factors that inhibit the transcription of PC-LECTIN throughinterfering with PC-LECTIN transcriptional activation may also be usefulfor the treatment of cancers expressing PC-LECTIN. Similarly, factorsthat are capable of interfering with PC-LECTIN processing may be usefulfor the treatment of cancers expressing PC-LECTIN. Cancer treatmentmethods utilizing such factors are also within the scope of theinvention.

General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies may be used for deliveringtherapeutic polynucleotide molecules to tumor cells synthesizingPC-LECTIN (i.e., antisense, ribozyme, polynucleotides encodingintrabodies and other PC-LECTIN inhibitory molecules). A number of genetherapy approaches are known in the art. Recombinant vectors encodingPC-LECTIN antisense polynucleotides, ribozymes, factors capable ofinterfering with PC-LECTIN transcription, and so forth, may be deliveredto target tumor cells using such gene therapy approaches.

The above therapeutic approaches may be combined with any one of a widevariety of chemotherapy or radiation therapy regimens. These therapeuticapproaches may also enable the use of reduced dosages of chemotherapyand/or less frequent administration, particularly in patients that donot tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, may beevaluated using various in vitro and in vivo assay systems. In vitroassays for evaluating therapeutic potential include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of PC-LECTIN to abinding partner, etc.

In vivo, the effect of a PC-LECTIN therapeutic composition may beevaluated in a suitable animal model. For example, xenogenic prostatecancer models wherein human prostate cancer explants or passagedxenograft tissues are introduced into immune compromised animals, suchas nude or SCID mice, are appropriate in relation to prostate cancer andhave been described (Klein et al., 1997, Nature Medicine 3: 402-408).For example, PCT Patent Application WO98/16628, Sawyers et al.,published Apr. 23, 1998, describes various xenograft models of humanprostate cancer capable of recapitulating the development of primarytumors, micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy may be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like. See, also, the Examples below.

In vivo assays that qualify the promotion of apoptosis may also beuseful in evaluating potential therapeutic compositions. In oneembodiment, xenografts from bearing mice treated with the therapeuticcomposition may be examined for the presence of apoptotic foci andcompared to untreated control xenograft-bearing mice. The extent towhich apoptotic foci are found in the tumors of the treated miceprovides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods may be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isnon-reactive with the patient's immune system. Examples include, but arenot limited to, any of a number of standard pharmaceutical carriers suchas sterile phosphate buffered saline solutions, bacteriostatic water,and the like (see, generally, Remington's Pharmaceutical Sciences ¹⁶thEdition, A. Osal., Ed., 1980).

Therapeutic formulations may be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations may be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancerand will generally depend on a number of other factors appreciated inthe art.

Cancer Vaccines

The invention further provides cancer vaccines comprising a PC-LECTINprotein or fragment thereof, as well as DNA based vaccines. In view ofthe tumor-restricted expression of PC-LECTIN, PC-LECTIN cancer vaccinesare expected to be effective at specifically preventing and/or treatingPC-LECTIN expressing cancers without creating non-specific effects onnon-target tissues. The use of a tumor antigen in a vaccine forgenerating humoral and cell-mediated immunity for use in anti-cancertherapy is well known in the art and has been employed in prostatecancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995,Int. J. Cancer 63: 231-237; Fong et al., 1997, J. Immunol. 159:3113-3117). Such methods can be readily practiced by employing aPC-LECTIN protein, or fragment thereof, or a PC-LECTIN-encoding nucleicacid molecule and recombinant vectors capable of expressing andappropriately presenting the PC-LECTIN immunogen.

For example, viral gene delivery systems may be used to deliver aPC-LECTIN-encoding nucleic acid molecule. Various viral gene deliverysystems that can be used in the practice of this aspect of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658-663).Non-viral delivery systems may also be employed by using naked DNAencoding a PC-LECTIN protein or fragment thereof introduced into thepatient (e.g., intramuscularly) to induce an anti-tumor response. In oneembodiment, the full-length human PC-LECTIN cDNA may be employed.

In one embodiment, a PC-LECTIN cancer vaccine is based on theidentification of immunogenic peptides within the PC-LECTIN amino acidsequence shown in FIG. 1A-D (SEQ ID NO: 2). As discussed further in theexamples below, specific portions of PC-LECTIN have been shown to induceT and B cell responses. The extracellular domain of PC-LECTIN (aminoacids 22-213 of FIG. 1A-D; SEQ ID NO: 2) has been used to generate animmune response in mice for the production of monoclonal antibodies; andpeptides within this domain, GLWRNGDGQTSGAC (SEQ ID NO: 25),GGPYLYQWNDDRCNM (SEQ ID NO: 26), EARLACESEGGVLL (SEQ ID NO: 27), havebeen used to generate an immune response in rabbits for the productionof polyclonal antibodies. Thus, these specific portions of PC-LECTIN,and polynucleotides encoding these portions, may be selected for theproduction of a cancer vaccine.

In another embodiment, PC-LECTIN nucleic acid molecules encodingspecific cytotoxic T lymphocyte (CTL) epitopes may be employed. CTLepitopes can be determined using specific algorithms (e.g., Epimer,Brown University) to identify peptides within a PC-LECTIN protein thatare capable of optimally binding to specified HLA alleles. One suitablealgorithm is the HLA Peptide Motif Search algorithm available at theBioinformatics and Molecular Analysis Section (BIMAS) web site. Thisalgorithm is based on binding of specific peptide sequences in thegroove of HLA Class I molecules and specifically HLA-A2 (Falk et al.,1991, Nature 351:290-6; Hunt et al., 1992, Science 255:1261-3; Parker etal., 1992, J. Immunol. 149:3580-7; Parker et al., 1994, J. Immunol.152:163-75). The HLA Peptide Motif Search algorithm allows location andranking of 8-mer, 9-mer, and 10-mer peptides from a complete proteinsequence for predicted binding to HLA-A2 as well as other Class Imolecules. Most HLA-A2 binding peptides are 9-mers, favorably containinga leucine at position 2 and a valine or leucine at position 9 (Parker etal., 1992, J. Immunol. 149:3580-7).

As discussed in the Examples below, predicted binding peptides forPC-LECTIN include WIGFTYKTA, ATGEHQAFT, FGNCVELQA, NCVELQASA, andDNHGFGNCV (SEQ ID NO: 6-10, respectively). Actual binding of peptides toHLA-A2 can be evaluated by stabilization of HLA-A2 expression on theantigen processing defective cell line T2 (Xue et al., 1997, Prostate30:73-8; Peshwa et al., 1998, Prostate 36:129-38). Immunogenicity ofspecific peptides can be evaluated in vitro by stimulation of CD8+CTL inthe presence of dendritic cells (Xue et al.; Peshwa et al., supra).

Various ex vivo strategies may also be employed. One approach involvesthe use of dendritic cells to present PC-LECTIN antigen to a patient'simmune system. Dendritic cells express MHC class I and II, B7co-stimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28: 65-69; Murphy et al.,1996, Prostate 29: 371-380). Dendritic cells can be used to presentPC-LECTIN peptides to T cells in the context of MHC class I and IImolecules. In one embodiment, autologous dendritic cells are pulsed withPC-LECTIN peptides capable of binding to MHC molecules. In anotherembodiment, dendritic cells are pulsed with the complete PC-LECTINprotein. Yet another embodiment involves engineering the overexpressionof the PC-LECTIN gene in dendritic cells using various implementingvectors known in the art, such as adenovirus (Arthur et al., 1997,Cancer Gene Ther. 4: 17-25), retrovirus (Henderson et al., 1996, CancerRes. 56: 3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57: 2865-2869), andtumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells expressing PC-LECTIN may also be engineered to expressimmune modulators, such as GM-CSF, and used as immunizing agents.

Anti-idiotypic anti-PC-LECTIN antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga PC-LECTIN protein. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can readily be adapted togenerate anti-idiotypic anti-PC-LECTIN antibodies that mimic an epitopeon a PC-LECTIN protein (see, for example, Wagner et al., 1997, Hybridoma16: 33-40; Foon et al., 1995, J Clin Invest 96: 334-342; Herlyn et al.,1996, Cancer Immunol Immunother 43: 65-76). Such an anti-idiotypicantibody can be used in cancer vaccine strategies.

Genetic immunization methods may be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing PC-LECTIN. Constructs comprising DNA encoding aPC-LECTIN protein/immunogen and appropriate regulatory sequences may beinjected directly into muscle or skin of an individual, such that thecells of the muscle or skin take-up the construct and express theencoded PC-LECTIN protein/immunogen. Expression of the PC-LECTIN proteinimmunogen results in the generation of prophylactic or therapeutichumoral and cellular immunity against prostate, breast, bladder, lung,bone, colon, pancreatic, testicular, cervical and ovarian cancers.Various prophylactic and therapeutic genetic immunization techniquesknown in the art may be used.

Kits

For use in the diagnostic and therapeutic applications described orsuggested above, kits are also provided by the invention. Such kits maycomprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe that is or can be detectably labeled. Suchprobe may be an antibody or polynucleotide specific for a PC-LECTINprotein or a PC-LECTIN gene or message, respectively. Where the kitutilizes nucleic acid hybridization to detect the target nucleic acid,the kit may also have containers containing nucleotide(s) foramplification of the target nucleic acid sequence and/or a containercomprising a reporter-means, such as a biotin-binding protein, such asavidin or streptavidin, bound to a reporter molecule, such as anenzymatic, florescent, or radioisotope label.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse. A label may be present on the on the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above.

The PC-LECTIN cDNA was deposited under the terms of the Budapest Treatyon Mar. 10, 1999, with the American Type Culture Collection (ATCC; 10801University Blvd., Manassas, Va. 20110-2209 USA) as plasmid p58P1D12-2,and has been assigned Accession No. 207152.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of cDNA Fragment of the PC-LECTIN Gene

Materials and Methods

LAPC Xenografts:

LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) andgenerated as described (Klein et al, 1997, Nature Med. 3: 402-408; Craftet al., 1999, Cancer Res. 59: 5030-5036). Androgen dependent andindependent LAPC-4 xenografts (LAPC-4 AD and AI, respectively) andLAPC-9 xenografts (LAPC-9 AD and AI, respectively) were grown in intactmale SCID mice or in castrated males, respectively, and were passaged assmall tissue chunks in recipient males. LAPC-4 AI xenografts werederived from LAPC-4 AD tumors and LAPC-9 AI xenografts were derived fromLAPC-9 AD tumors. To generate the AI xenografts, male mice bearing LAPCAD tumors were castrated and maintained for 2-3 months. After the LAPCtumors re-grew, the tumors were harvested and passaged in castratedmales or in female SCID mice.

Cell Lines:

Human cell lines (e.g., HeLa) were obtained from the ATCC and weremaintained in DMEM with 10% fetal calf serum.

RNA Isolation:

Tumor tissue and cell lines were homogenized in Trizol reagent (LifeTechnologies, Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides:

DPNCDN (cDNA synthesis primer) (SEQ ID NO: 11): 5′TTTTGATCAAGCTT₃₀3′Adaptor 1 (SEQ ID NO: 12 and 13, respectively):5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′                              3′GGCCCGTCCTAG5′ Adaptor 2 (SEQ ID NO: 14and 15, respectively): 5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCGGAG3′                              3′CGGCTCCTAG5′ PCR primer 1 (SEQ ID NO:16): 5′CTAATACGACTCACTATAGGGC3′ Nested primer (NP)1 (SEQ ID NO: 17):5′TCGAGCGGCCGCCCGGGCAGGA3′ Nested primer (NP)2 (SEQ ID NO: 18):5′AGCGTGGTCGCGGCCGAGGA3′

Suppression Subtractive Hybridization:

Suppression subtractive hybridization (SSH) was used to identify cDNAscorresponding to genes that may be up-regulated in androgen dependentprostate cancer compared to androgen independent cancer.

Double stranded cDNAs corresponding to the LAPC-9 AD xenograft (tester)and the LAPC-9 AI tissue (driver) were synthesized from 2 μg of poly(A)′RNA isolated from the xenografts, as described above, using CLONTECH'sPCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN asprimer. First and second-strand synthesis were carried out as describedin the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1,Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3hrs. at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1)and ethanol precipitated.

Driver cDNA (LAPC-9AI) was generated by combining in a 1 to 1 ratio DpnII digested LAPC-9AI cDNA with a mix of digested cDNAs from BPH tissueand human cell lines HeLa, 293, A431, Colo 205 and mouse liver, in orderto ensure that murine genes were subtracted from the tester cDNA (LAPC-9AD).

Tester cDNA (LAPC-9 AD) was generated by diluting 1 μl of Dpn IIdigested LAPC-9 AD cDNA (400 ng) in 5 μl of water. The diluted cDNA (2μl, 160 ng) was then ligated to 2 μl of adaptor 1 and adaptor 2 (10 μM),in separate ligation reactions, in a total volume of 10 μl at 16° C.overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation wasterminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) adaptor 1-and adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlayed with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, 72° C. for 1.5 minutes. The PCR products wereanalyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid! culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs were generated from 1 μg of mRNA with oligo (dT)12-18priming using the Gibco-BRL Superscript Preamplification system. Themanufacturers protocol was used and included an incubation for 50 min at42° C. with reverse transcriptase followed by RNAse H treatment at 37°C. for 20 min. After completing the reaction, the volume was increasedto 200 μl with water prior to normalization. First strand cDNAs from 16different normal human tissues were obtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:19) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 20) to amplifyβ-actin. First strand cDNA (5 μl) was amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction was removed at 18,20, and 22 cycles and used for a0arose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: initial denaturation was at 94° C. for 15 sec, followed by a18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 bp β-actinbands from multiple tissues were compared by visual inspection. Dilutionfactors for the first strand cDNAs were calculated to result in equalβ-actin band intensities in all tissues after 22 cycles of PCR. Threerounds of normalization were required to achieve equal band intensitiesin all tissues after 22 cycles of PCR.

To determine expression levels of the PC-LECTIN gene, 5 μl of normalizedfirst strand cDNA was analyzed by PCR using 25, 30, and 35 cycles ofamplification using the following primer pairs, which were designed withthe assistance of (MIT) (SEQ ID NO: 21 and 22, respectively):

58P1D12.1 5′ CCTGCTTCAGTAACAACCACATTCT 3′ 58P1D12.2 5′CTTTACCAGTGGAATGATGACAGG 3′

Semi quantitative expression analysis was achieved by comparing the PCRproducts at cycle numbers that give light band intensities.

Results

Several SSH experiments were conduced as described in the Materials andMethods, supra, and led to the isolation of numerous candidate genefragment clones. All candidate clones were sequenced and subjected tohomology analysis against all sequences in the major public gene and ESTdatabases in order to provide information on the identity of thecorresponding gene and to help guide the derision to analyze aparticular gene for differential expression. In general, gene fragmentswhich had no homology to any known sequence in any of the searcheddatabases, and thus considered to represent novel genes, as well as genefragments showing homology to previously sequenced expressed sequencetags (ESTs), were subjected to differential expression analysis byRT-PCR and/or northern analysis.

One of the cDNA clones, designated 58P1D12, was 427 bp in length andshowed weak homology to an EST derived from pig muscle as well assignificant homology to hamster layilin, a cell surface molecule withhomology to C-type lectins. The SSH fragment contained an ORF of 129amino acids, which showed significant homology to layilin. The ORF ofthis fragment corresponds to the central region of layilin and containsthe transmembrane domain. The full length cDNA encoding the 58P1D12 genewas subsequently isolated using this cDNA and structurally analyzed(Example 2, below) and re-named PC-LECTIN.

Differential expression analysis by RT-PCR using primers derived fromthe PC-LECTIN SSH clone showed that the 58P1D12/PC-LECTIN gene isessentially expressed in normal testis and in the prostate tumorxenografts examined (FIG. 3). At higher cycles of amplification (i.e.,30+), lower level expression was detected in prostate, spleen andplacenta. Northern blot analysis using the full length PC-LECTIN cDNA asa probe (see Example 3, below) showed expression of 1.8 and 3.0 kbtranscripts only in normal testis and in LAPC9 AD RNA (FIG. 4). Lowerexpression levels are detected in LAPC-4AD, LAPC-4AI and LAPC-9 AI.

Example 2 Isolation of Full Length PC-LECTIN Encoding cDNA

The 427 bp 58P1D12/PC-LECTIN gene fragment (Example 1) was used toisolate 30 additional cDNAs encoding the PC-LECTIN gene. A full lengthcDNA clone for PC-LECTIN was isolated from an LAPC-9 AD library. ThecDNA (clone 2) is 25510 bp in length and encodes a 273 amino acid ORF.Analysis of the ORF identifies an N-terminal signal sequence and atransmembrane domain that indicate PC-LECTIN to be a type 1atransmembrane protein with the N-terminus on the outside and acytoplasmic C-terminus. The full length PC-LECTIN cDNA has beendeposited with the American Type Culture Collection (“ATCC”) (Mannassas,Va.) as plasmid p58P1D12-2 on Mar. 10, 1999 as ATCC Accession Number207152. The PC-LECTIN cDNA clone therein can be excised therefrom usingEcoRIIXbaI double digest (EcoRI at the 5′end, XbaI at the 3′end).

Amino acid alignment of the PC-LECTIN sequence with hamster layilinindicates a relationship to layilin (FIG. 2A). However, PC-LECTIN doesnot exhibit the talin association domain, suggesting that PC-LECTIN doesnot interact with the cytoskeleton in the same manner as layilin. Otherstructural differences are also apparent (FIG. 2A). Alignment of the2550 bp PC-LECTIN cDNA with the 1747 bp cDNA of hamster layilin cDNAshows homology over a 591 bp region (FIG. 2B). The rest of the PC-LECTINregion is significantly different from layilin, which is reflected inthe differences in the amino acid sequence of the C-terminal half of theextracellular domain and the entire cytoplasmic domain. This suggeststhat while PC-LECTIN and layilin are related and probably constitute asub-family of lectins, PC-LECTIN is unlikely to be the human form oflayilin.

Example 3 PC-LECTIN Gene Expression Analysis

Initial analysis of PC-LECTIN mRNA expression in normal human tissueswas conducted by northern blotting two multiple tissue blots obtainedfrom Clontech (Palo Alto, Calif.), comprising a total of 16 differentnormal human tissues using labeled PC-LECTIN cDNA as a probe. RNAsamples were quantitatively normalized with a β-actin probe. The resultsare shown in FIG. 4 (Panels A and B). Expression was only detected innormal testis. These northern blots showed two transcripts ofapproximately 1.8 kb and 3.0 kb.

This initial analysis was extended by using the PC-LECTIN probe toanalyze an RNA dot blot matrix of 50 normal human tissues (Clontech,Palo Alto, Calif.; Human Master Blot™). The results show strongPC-LECTIN expression only in testis and fetal spleen (FIG. 14). Lowerlevels of expression were detected in salivary gland and fetal kidney.No expression was detected in the following tissues: brain, amygdala,caudate nucleus, cerebellum, cerebral cortex, frontal lobe, hippocampus,medulla oblongata, occipital lobe, putamen, substantia nigra, temporallobe, thalamus, sub-thalamic nucleus, spinal cord, heart, aorta,skeletal muscle, colon, bladder, uterus, prostate, stomach, ovary,pancreas, pituitary gland, adrenal gland, thyroid gland, mammary gland,kidney, liver, small intestine, spleen, thymus, peripheral leukocytes,lymph node, bone marrow, appendix, lung, trachea, placenta, fetal brain,fetal heart, fetal liver, fetal thymus fetal lung.

To analyze PC-LECTIN expression in human prostate cancer tissues, RNAsderived from human prostate cancer xenografts were also analyzed. AllRNA samples were quantitatively normalized by ethiduim bromide stainingand subsequent analysis with a labeled β-actin probe. The results (FIG.4C) show high level PC-LECTIN expression, particularly in the LAPC-9 ADxenograft, with lower but significant level expression detected in theremaining xenografts.

Northern blot analysis using a PC-LECTIN SSH fragment probe shows thatPC-LECTIN is highly expressed in tumors that are grown eithersubcutaneously (sc; FIG. 5; Lane 2) or intratibially (it; FIG. 5;Lane 1) within the mouse bone. To investigate whether PC-LECTINexpression is dependent on the presence of androgen, LAPC-9 AD tumorswere grown in male SCID mice. The mice were castrated and tumors wereharvested 28 days later. The expression of PC-LECTIN in tumors of 28day-post castrated males was compared to the expression in tumors ofintact males. The results show that PC-LECTIN expression is dramaticallyreduced in tumors from castrated males (FIG. 6). As a control,expression of a known androgen-regulated gene, TMPRSS2 (See WO99/62942),was also shown to be down-regulated after castration (FIG. 6). Thesedata suggest that PC-LECTIN expression in prostate tumors is dependenton the presence of androgen.

In addition, RT-PCR can be used to analyze expression of PC-LECTIN invarious tissues, including patient-derived cancers. First strand cDNAsare generated from 1 μg of mRNA with oligo (dT)12-18 priming using theGibco-BRL Superscript Preamplification system. The manufacturersprotocol can be used and includes an incubation for 50 min at 42° C.with reverse transcriptase followed by RNAse H treatment at 37° C. for20 min. After completing the reaction, the volume is increased to 200 μlwith water prior to normalization. First strand cDNAs are prepared fromvarious tissues of interest. Normalization can be performed by PCR usingprimers to actin and GAPDH. Semi-quantitative PCR is performed usingprimers to PC-LECTIN.

Example 4 Biochemical Characterization of PC-LECTIN Protein

To initially characterize the PC-LECTIN protein, PC-LECTIN cDNA wascloned into the pcDNA 3.1 Myc-His plasmid (Invitrogen), which encodes a6His tag at the carboxyl terminus, transfected into 293T cells, andlabeled with a water soluble biotinylation reagent that is excluded fromlive cells. Biotinylated cell surface proteins were affinity purifiedwith streptavidin-sepharose and probed with anti-His antibodies. Westernblotting of streptavidin purified proteins clearly show cell surfacebiotinylation of PC-LECTIN in transfected 293T cells (FIG. 7). PC-LECTINprotein was not detected in streptavidin precipitates fromnon-biotinylated transfected cells (FIG. 7).

Example 5 Expression of Recombinant PC-LECTIN Protein in MammalianSystems

For mammalian expression, PC-LECTIN may be cloned into several vectors,including pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralexpression vector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Usingthese expression vectors, PC-LECTIN may be expressed in several celllines, including PC-3, NIH 3T3, mouse L cell fibroblasts and 293T.

Recombinant retrovirus encoding the PC-LECTIN protein was generated inhuman 293T cells (Pear et al., 1993, PNAS 90:8392-8396) and was used toinfect NIH 3T3 cells, which were selected in G418 for two weeks togenerate stable lines. Expression of PC-LECTIN was confirmed by northernblotting using a PC-LECTIN cDNA probe.

The mammalian cell lines expressing PC-LECTIN may be used in several invitro and in vivo assays, including cell proliferation in tissueculture, activation of apoptotic signals, tumor formation in SCID mice,and in vitro invasion using a membrane invasion culture system (MICS)(Welch et al., Int. J. Cancer 43: 449-457).

Example 6 Production of Recombinant PC-LECTIN in a Baculovirus System

To generate a recombinant PC-LECTIN protein in a baculovirus expressionsystem, the PC-LECTIN cDNA is cloned into the baculovirus transfervector pBlueBac 4.5 (Invitrogen), which provides a His-tag at theN-terminus Specifically, pBlueBac-PC-LECTIN is co-transfected withhelper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda)insect cells to generate recombinant baculovirus (see Invitrogeninstruction manual for details). Baculovirus is then collected from cellsupernatant and purified by plaque assay.

Recombinant PC-LECTIN protein is then generated by infection of HighFiveinsect cells (InVitrogen) with the purified baculovirus. RecombinantPC-LECTIN protein may be detected using anti-PC-LECTIN antibody.PC-LECTIN protein may be purified and used in various cell based assaysor as immunogen to generate polyclonal and monoclonal antibodiesspecific for PC-LECTIN.

Example 7 Generation of a Secreted Recombinant PC-LECTIN-AlkalinePhosphatase Fusion Protein

The identification of proteins interacting with PC-LECTIN could helpassign function and may identify novel therapeutic targets anddiagnostic markers for prostate cancer. The construction of an alkalinephosphatase-PC-LECTIN fusion protein may be used to detect and cloneproteins interacting with PC-LECTIN while also generating an immunogenfor monoclonal antibody and polyclonal antibody production.

The AP-TAG system from GenHunter Corporation (Nashville, Tenn., cat#Q202) was utilized to make the fusion protein and for detection ofPC-LECTIN binding. The PC-LECTIN cDNA (FIG. 1A-D; SEQ ID NO: 1), withoutthe signal sequence, was cloned into pAPtag-5 (GenHunter Corp.Nashville, Tenn.). The PC-LECTIN.HindIII and PC-LECTIN.BamHI primersshown below were used to amplify the PC-LECTIN open reading frame fromamino acids 22 to 213 from the plasmid template PC-LECTIN clone 2. TheHindIII and BamHI digested PCR product was ligated into HindIII andBgIII digested pAPtag-5, while keeping the IgGK signal sequence,PC-LECTIN ORF, and alkaline phosphatase all in frame. The PC-LECTIN-APfusion protein contains an IgGK signal sequence to promote secretionalong with myc/His tags at the carboxy terminus of alkaline phosphatase.

PC-LECTIN.HINDIII Primer (SEQ ID NO: 23): GTGTAAGCTTCCCGCCGCGTGGTCAGCGGCPC-LECTIN.BAMHI Primer (SEQ ID NO: 24): CACAGGATCCTATACCTGCTTCAGTAAC

This PC-LECTIN-AP fusion protein construct was used to transfect 293Tcells, and the presence of secreted fusion protein into the culturemedia was monitored by western blot using anti-alkaline phosphatase andanti-HIS antibodies. The results of this analysis, shown in FIG. 8, showdetection of an approximately 100 kDa fusion protein in conditionedmedia of transfected 293T cells.

Amino acids 22 to 213 were also cloned into the pAPTag-5 vector usingPCR with primers containing restriction enzymes HindIII and XhoI toproduce a IgGK signal sequence fusion at the N-terminus and the myc/Histags at the C-terminus of PC-LECTIN extracellular domain. This constructis similar to 58P1D12pAPtag above but without the AP fusion.

The entire coding sequence of PC-LECTIN (aa 1-273) was cloned into pSRa.Primers encoding the ORF and the restriction sites EcoRI and XbaIamplified the insert from PC-LECTIN clone 2 (pBK.CMV). The insert wasligated to pSRa after digestion of both with EcoRI and XbaI. Thisconstruct was used to generate virus and make cell lines stablyexpressing PC-LECTIN protein.

The entire coding sequence of PC-LECTIN (aa 1-273) was cloned intopcDNA3.1/myc-HIS (Invitrogen). Primers encoding the ORF and therestriction sites EcoRI and XbaI amplified the insert from PC-LECTINclone 2 (pBK.CMV). The insert was ligated to pcDNA3.1/myc-HIS(Invitrogen) after digestion of both with EcoRI and XbaI. Western blotanalysis confirmed expression PC-LECTIN protein when 293T cells weretransfected with this construct.

Example 8 Detection and Cloning of PC-LECTIN Binding Partner

PC-LECTIN is a transmembrane protein with lectin C-type domains that mayinteract with a binding partner protein. To detect PC-LECTIN receptorbinding, several cell lines, tissues, and plates coated withglycoprotein (e.g., human or mouse IgG, bovine RNase, ovalbumin, humantransferrin, fetuin glycophorin, sialogylcophorin) are incubated withthe PC-LECTIN-AP fusion protein using procedures in Cheng and Flanagan,1994, Cell 79:157-168. After washing the cells and adding the APsubstrate BCIP, which forms an insoluble blue precipitate upondephosphorylation, PC-LECTIN binding to cell surface receptor can bedetected using a microscope to look for cells staining blue. The celllines that may be screened include LNCaP, PC-3, DU145, TSUPR, PREC,LAPC4, 293T, NIH 3T3, and other cancer cell lines. Tissues may also bescreened such as the LAPC xenografts, prostate tissue and prostatecarcinoma. Once PC-LECTIN-AP cell surface binding is observed, anequilibrium dissociation rate constant can be calculated to evaluate thestrength of the binding interaction. In addition, the number of cellsurface receptors per cell can be determined. The cell line or tissuewith the highest binding capacity for PC-LECTIN may then be used toclone the receptor. Binding of PC-LECTIN to a specific carbohydratemoiety can be confirmed by demonstrating binding inhibition by lowconcentrations of specific related monosaccharide.

Expression cloning strategies such as those described in Tartaglia etal., 1995, Cell 83: 12631271, Cheng and Flanagan and others may be usedto clone the receptor for PC-LECTIN. In one approach, an expressionlibrary is constructed from the cells showing PC-LECTIN-AP binding. Thelibrary is made as pools of approximately 1000 clones and is screened bya sib selection procedure. Transient transfection of COS cells with DNAfrom each pool and subsequent screening with PC-LECTIN-AP binding,washing, and staining for AP activity identifies cells binding PC-LECTINand consequently expression of PC-LECTIN receptors. After successiverounds of pool subdivision and screening, single colonies binding toPC-LECTIN-AP are identified.

Alternatively, an expression library is generated in phage usingstandard technology (Stone J. in Current Protocols in Molecular Biology(1997): 20.3.1-20.3.9). Membrane lifts are probed using the PC-LECTIN-APfusion protein of Example 6 and a BCIP alkaline phosphatase assay isused for detection. Plaques binding PC-LECTIN-AP and producing a blueprecipitate are picked and plasmids containing the gene for the receptorare excised. An important advantage of this approach is that cytoplasmicor secreted proteins interacting with PC-LECTIN are also identified.

Example 9 Expression and Purification of PC-LECTIN Extracellular Domain

293T cells were transfected with a Tag5 secretion expression vectorencoding the extracellular domain (amino acids 22-213) of PC-LECTIN witha C-terminal 6×His tag. A stable cell line was then generated by zeocinselection. The cell line was grown in spinner culture in 293 SFMII serumfree medium (Gibco) and conditioned medium was collected forpurification. Conditioned medium was concentrated and buffer exchangedinto binding buffer (50 mM sodium phosphate buffer pH 8.0, 500 mM NaCl,and 10 mM imidazole) and subjected to immobilized metal affinitychromatography using NI-NTA agarose (Qiagen). The starting conditionedmedium, the flow through, and the eluted purified material was run on a10-20% SDS-PAGE gel and silver stained (FIG. 9A) or transferred tonitrocellulose and subjected to western blotting using an anti-His pAb(FIG. 9B).

Example 10 Polyclonal and Monoclonal Antibodies to PC-LECTIN

To generate polyclonal antibodies towards PC-LECTIN, three differentpeptides were generated towards the extracellular domain of PC-LECTIN.The peptide sequences are:

GLWRNGDGQTSGAC, (14mer; SEQ ID NO: 25) GGPYLYQWNDDRCNM, (15mer; SEQ IDNO: 26) and EARLACESEGGVLL. (14mer; SEQ ID NO: 27)

The peptides were conjugated to KLH (keyhole limpet hemocyanin) and wereused to immunize rabbits. Serum from the rabbits was tested forreactivity towards PC-LECTIN protein using western blotting of celllysates and using FACS on whole cells (See Example 11 below). Titer wasmonitored by ELISA to the peptide and by western blotting usingrecombinant cell lines expressing the PC-LECTIN cDNA. Subsequentexperiments were performed with antibodies generated from amino acids204-217 of the PC-LECTIN protein (GLWRNGDGQTSGAC; SEQ ID NO: 25).

To generate monoclonal antibodies, the extracellular domain of PC-LECTINwas efficiently expressed and purified from conditioned media of 293Tcells expressing the Tag5 PC-LECTIN secretion vector as described inExample 9 above. The purified protein was used to immunize BalbC mice.Mice were initially injected intraperitoneally with 50 μg of protein incomplete Freund's adjuvant and then boosted 3 weeks later with 50 μgprotein in incomplete Freund's adjuvant. Boosts then continued on a 2week immunization schedule and titers of immunized mouse serum weremonitored by ELISA using Tag5 PC-LECTIN as target and specificity bywestern blot analysis of cell lines and tissue lysates.

Example 11 PC-LECTIN Expression in Recombinant Cell Lines and Testis

The immunized mouse serum was used to analyze PC-LECTIN expression bywestern blot and immunoprecipitation using cell lysates of recombinantcell lines and normal testis. In addition, PC-LECTIN expression on thecell surface of Rat1-PC-LECTIN cells was analyzed by flow cytometryusing the immunized mouse serum.

Rat1 cells stably infected with either neo control virus or virusencoding PC-LECTIN were lysed in RIPA buffer (25 mM Tris pH 7.5, 150 mMNaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA,100 μg/ml PMSF, and 2 μM leupeptin). 200 μg of lysates were thensubjected to immunoprecipitation with serum from mice immunized withpurified Tag5-PC-LECTIN protein. Briefly, 3 μl of serum was incubatedwith lysates (200 μg protein in 1 ml) and incubated overnight at 4° C.50 μl of a 50% slurry of Protein G beads in RIPA buffer was then addedand further incubated for 1 hour at room temperature. Immunoprecipitateswere washed 4× with RIPA buffer and solubilized in 40 μl of 3×SDS-PAGEsample buffer and heated at 100° C. 25 μl of solubilizedimmunoprecipitates or 25 μg of the indicated RIPA lysates were separatedon a 10-20% SDS-PAGE gel and transferred to nitrocellulose.

Western blot analysis was then carried out with either an affinitypurified rabbit anti-PC-LECTIN peptide pAb (2 μg/ml, FIG. 10A) or with a1:1000 dilution of immunized mouse serum diluted into Tris bufferedsaline containing 0.15% Tween-20 (TBS-T, pH 7.5) and 1% nonfat milk(FIG. 10B). Blots were incubated for 2 hours at room temperature withserum and then washed 3× with TBS-T. Immunoreactive bands were thendeveloped by incubation with either anti-rabbit Ig or anti-mouse IgGHRP-conjugated secondary Abs and visualized by incubation with enhancedchemiluminescence substrate (ECL, Amersham) and exposure toautoradiographic film.

Cell lysates of 293T cells transiently transfected with either pCDNA3.1Myc/His PC-LECTIN or empty vector and of Rat1 cells stably infected witheither neo control or PC-LECTIN retrovirus and of normal testis wereseparated by SDS-PAGE and transferred to nitrocellulose. Westernanalysis was then carried out as described above. The results are shownin FIG. 11. Indicated with arrows are the 47 kD band representing fulllength PC-LECTIN, the 40 kD extracellular domain, and the 55 kD Myc/Histagged protein.

Cell surface recognition of PC-LECTIN on Rat1 cells with Tag5 PC-LECTINimmunized mouse serum was analyzed by flow cytometry. Either Rat1-neo orRat1-PC-LECTIN cells (5×10⁵) were incubated with a 1:2000 dilution ofTag5 PC-LECTIN immunized mouse serum in PBS containing 1% FBS and 0.02%NaN3 (flow buffer) for 1 hour on ice. Cells were washed 2× with ice coldflow buffer and then incubated with a 1:200 dilution of anti-mouseIgG-FITC conjugate on ice for 30 minutes. Cells were washed 2× with flowbuffer and resuspended in PBS containing 1% paraformaldehyde. 3,000cells from each sample were then analyzed by flow cytometry for cellsurface staining of PC-LECTIN. Results are shown in FIG. 12.

Cell surface expression of PC-LECTIN was further analyzed usingimmunohistochemical analysis of formalin-fixed, paraffin-embedded cellpellets. The PC-LECTIN-transfected 293T cells were labeled with rabbitpolyclonal antibody at 7.5 μg/ml (SHIER II pretreatment). Cell surfaceexpression of PC-LECTIN was detected as shown in FIG. 13. The antibodydid not stain parental 293T cells.

Example 12 Carbohydrate Binding Specificity of PC-LECTIN

PC-LECTIN was analyzed for carbohydrate binding specificity using a96-well microassay employing the Tag5 extracellular domain of PC-LECTINpurified from conditioned medium. Analysis of the ability of PC-LECTINto bind to a variety of carbohydrate moieties on purified proteinpreparations demonstrates a specificity for high mannose residues aswell as N-acetylglucosamine. This specificity is similar to that seenfor the lectin Concanavalin A.

The wells of a 96-well microtiter plate were coated with the appropriateglycoprotein at 1 ug/well in PBS and incubated overnight at 37° C. Thewells were washed once with 1× Tris-Buffered Saline (TBS) and thenblocked with 3% BSA (Sigma) in PBS for 1 hour with rocking. The wellswere incubated with either buffer control, or 50 ng of PC-LECTIN or 50ng of Concanavalin A (ConA) in 1×TBS supplemented with 2 mM CaCl₂. Theplates were then incubated for 2 hours at room temperature with rocking.The plates were then washed 3× with TBS, 2mM CaCl₂, 0.05% Tween-20, andonce with TBS, 2 mM CaCl₂. The wells were then incubated for 1 hour atroom temperature with either an anti-His6 rabbit pAb (for PC-LECTINdetection, Santa Cruz Biotechnology) or an anti-ConA rabbit pAb (forConA detection, Vector Laboratories) each diluted 1/1000 in TBS, 2 mMCaCI₂ plus 1% BSA. The wells were washed as before. The wells were thenincubated with anti-rabbit Ig HRP conjugate diluted 1/3,000 with TBS, 2mM CaCl₂ plus 1% BSA. The wells were washed again and then developedusing TMB ELISA (GIBCO-BRL) of the manufacturer's guidelines, and theoptical density was measured at 450 nM. Data are shown in Table 1, andrepresent the means of duplicate determinations.

TABLE 1 PC-LECTIN carbohydrate binding specificity PC- LECTIN ConAGlycoprotein Sugar Type OD450 OD450 Chicken Egg Albumin High Mannose0.962 3.12 Avidin High Mannose 1.104 3.361 Chorionic GonadotropinN-Acetylneuraminic acid 0.013 — Chicken Egg conalbumin Bisecting GlcNAc0.663 3.1 Thyrogolobulin, Bovine Mannose-6-phosphate 0.979 — Laminin,Human tri-mannose core, 0.389 — GlcNAc C-7913 1 0.038 — T antigengalacto-N-biose, GalB1, — 0.015 3GalNAc —: not examined Con A:concanavalin A 1: C-7913 carboxyethylthioethyl2-acetoamido-2-deoxy-4-o-B-s-galactopyranosyl-b-d-glucopyranoside BSAGlcNAc: N-acetyl glucosamine GalNAc: N-acetyl galactosamine GalB1:galactose beta 1 linkage

Example 13 Predicted Binding of PC-LECTIN Peptides to HLA-A2

To identify PC-LECTIN peptides predicted to bind to the human MHC classI molecule HLA-A2, the complete amino acid sequence of the58P1D12(PC-LECTIN)-F3C4 family member protein was entered into the HLAPeptide Motif Search algorithm found in the Bioinformatics and MolecularAnalysis Section (BIMAS) Web site. The results of 58P1D12-F3C4 predictedbinding peptides are shown in Table 2. The top 5 ranking candidates areshown along with their location, the amino acid sequence of eachspecific peptide, and an estimated binding score. The binding scorecorresponds to the estimated half-time of dissociation of complexescontaining the peptide at 37° C. at pH 6.5. Peptides with the highestbinding score are predicted to be the most tightly bound to HLA Class Ion the cell surface and thus represent the best immunogenic targets forT-cell recognition. Actual binding of peptides to HLA-A2 can beevaluated by stabilization of HLA-A2 expression on theantigen-processing defective cell line T2 (Xue et al., 1997, Prostate30:73-8; Peshwa et al., 1998, Prostate 36:129-38). Immunogenicity ofspecific peptides can be evaluated in vitro by stimulation of CD8+cytotoxic T lymphocytes (CTL) in the presence of dendritic cells (Xue etal., 1997, Prostate 30:73-8; Peshwa et al., 1998, Prostate 36:129-38).

TABLE 2 Predicted Peptide Binding Scores Score (Estimate of StartSubsequence half time of disasso- Rank Position Residue Listing ciation)1 6 WIGFTYKTA 4.7 (SEQ ID NO: 28) 2 21 ATGEHQAFT 2.3 (SEQ ID NO: 29) 341 FGNCVELQA 0.2 (SEQ ID NO: 30) 4 43 NCVELQASA 0.1 (SEQ ID NO: 31) 5 37DNHGFGNCV 0.1 (SEQ ID NO: 32)

Example 14 Identification of Potential Signal Transduction Pathways

To determine whether PC-LECTIN directly or indirectly activates knownsignal transduction pathways in cells, luciferase (luc) basedtranscriptional reporter assays are carried out in cells expressingPC-LECTIN. These transcriptional reporters contain consensus bindingsites for known transcription factors that lie downstream of wellcharacterized signal transduction pathways. The reporters and examplesof their associated transcription factors, signal transduction pathways,and activation stimuli are listed below.

1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation

3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

4. ARE-luc, androgen receptor; steroids/MAPK;growth/differentiation/apoptosis

5. p53-luc, p53; SAPK; growth/differentiation/apoptosis

6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

PC-LECTIN-mediated effects may be assayed in cells showing mRNAexpression. Luciferase reporter plasmids may be introduced by lipidmediated transfection (TFX-50, Promega). Luciferase activity, anindicator of relative transcriptional activity, is measured byincubation of cells extracts with luciferin substrate and luminescenceof the reaction is monitored in a luminometer.

Example 15 In Vitro Assays of PC-LECTIN Function

The expression of PC-LECTIN in prostate cancer provides evidence thatthis gene has a functional role in tumor progression and/or tumorinitiation. It is possible that PC-LECTIN functions as a receptorinvolved in activating proliferation signals. PC-LECTIN function can beassessed in mammalian cells using in vitro approaches. For mammalianexpression, PC-LECTIN can be cloned into a number of appropriatevectors, including pcDNA 3.1 myc-His-tag and the retroviral vectorpSRαtkneo (Muller et al., 1991, MCB 11:1785). Using such expressionvectors, PC-LECTIN can be expressed in several cell lines, includingPC-3, NIH 3T3, LNCaP and 293T. Expression of PC-LECTIN can be monitoredusing anti-PC-LECTIN antibodies and northern blot analysis.

Mammalian cell lines expressing PC-LECTIN can be tested in several invitro and in vivo assays, including cell proliferation in tissueculture, activation of apoptotic signals, tumor formation in SCID mice,and in vitro invasion using a membrane invasion culture system (MICS;Welch et al.) Int. J. Cancer 43: 449-457). PC-LECTIN cell phenotype iscompared to the phenotype of cells that lack expression of PC-LECTIN.

Cell lines expressing PC-LECTIN can also be assayed for alteration ofinvasive and migratory properties by measuring passage of cells througha matrigel coated porous membrane chamber (Becton Dickinson). Passage ofcells through the membrane to the opposite side is monitored using afluorescent assay (Becton Dickinson Technical Bulletin #428) usingcalcein-Am (Molecular Probes) loaded indicator cells. Cell linesanalyzed include parental and PC-LECTIN overexpressing PC3, NIH 3T3 andLNCaP cells. To determine whether PC-LECTIN-expressing cells havechemoattractant properties, indicator cells are monitored for passagethrough the porous membrane toward a gradient of PC-LECTIN conditionedmedia compared to control media. This assay may also be used to qualifyand quantify specific neutralization of the PC-LECTIN induced effect bycandidate cancer therapeutic compositions.

The function of PC-LECTIN can be evaluated using anti-sense RNAtechnology coupled to the various functional assays described above,e.g. growth, invasion and migration. Anti-sense RNA oligonucleotides canbe introduced into PC-LECTIN expressing cells, thereby preventing theexpression of PC-LECTIN. Control and anti-sense containing cells can beanalyzed for proliferation, invasion, migration, apoptotic andtranscriptional potential. The local as well as systemic effect of theloss of PC-LECTIN expression can be evaluated.

Example 16 In Vivo Assay for PC-LECTIN Tumor Growth Promotion

The effect of the PC-LECTIN protein on tumor cell growth may beevaluated in vivo by gene overexpression in tumor-bearing mice. Forexample, SCID mice can be injected subcutaneously on each flank with1×10⁶ of either PC3, TSUPR1, or DU 145 cells containing tkNeo emptyvector or PC-LECTIN. At least two strategies may be used: (1)Constitutive PC-LECTIN expression under regulation of a promoter such asa constitutive promoter obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, provided such promoters arecompatible with the host cell systems, and (2) Regulated expressionunder control of an inducible vector system, such as ecdysone, tet,etc., provided such promoters are compatible with the host cell systems.Tumor volume is then monitored at the appearance of palpable tumors andfollowed over time to determine if PC-LECTIN expressing cells grow at afaster rate and whether tumors produced by PC-LECTIN-expressing cellsdemonstrate characteristics of altered aggressiveness (e.g. enhancedmetastasis, vascularization, reduced responsiveness to chemotherapeuticdrugs). Additionally, mice may be implanted with 1×10⁵ of the same cellsorthotopically to determine if PC-LECTIN has an effect on local growthin the prostate or on the ability of the cells to metastasize,specifically to lungs, lymph nodes, and bone marrow.

The assay is also useful to determine the PC-LECTIN inhibitory effect ofcandidate therapeutic compositions, such as for example, PC-LECTINintrabodies, PC-LECTIN antisense molecules and ribozymes.

Example 17 Western Analysis of PC-LECTIN Expression in SubcellularFractions

Sequence analysis of PC-LECTIN revealed the presence of two type-Clectin domains and a transmembrane domain. The cellular location ofPC-LECTIN can be assessed further using subcellular fractionationtechniques widely used in cellular biology (Storrie B, et al. MethodsEnzymol. 1990; 182:203-25). Prostate cell lines can be separated intonuclear, cytosolic and membrane fractions. The expression of PC-LECTINin the different fractions can be tested using western blottingtechniques.

Alternatively, to determine the subcellular localization of PC-LECTIN,293T cells can be transfected with an expression vector encodingHIS-tagged PC-LECTIN (PCDNA 3.1 MYC/HIS, Invitrogen). The transfectedcells can be harvested and subjected to a differential subcellularfractionation protocol as previously described (Pemberton, P. A. et al,1997, J of Histochemistry and Cytochemistry, 45:1697-1706.) Thisprotocol separates the cell into fractions enriched for nuclei, heavymembranes (lysosomes, peroxisomes, and mitochondria), light membranes(plasma membrane and endoplasmic reticulum), and soluble proteins.

Throughout this application, various publications are referenced. Thedisclosures of these publications are hereby incorporated by referenceherein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

1. A method of detecting the presence of a PC-LECTIN protein in a testsample, comprising: providing the test sample, wherein the test sampleis prostate tissue; contacting the sample with an antibody or antigenbinding fragment thereof which binds to a PC-LECTIN protein comprisingthe amino acid sequence of SEQ ID NO. 2; and detecting the binding ofPC-LECTIN protein in the sample thereto, wherein detection of thePC-LECTIN protein correlates to the presence of an androgen-dependentprostate cancer cell in the test sample.
 2. The method of claim 1,further comprising quantifying and comparing the amount of PC-LECTINprotein present in the test sample with the amount of PC-LECTIN proteinpresent in a comparable normal sample, the presence of a higher amountof PC-LECTIN protein in the test sample relative to the normal sampleindicating the presence of a prostate cancer expressing PC-LECTINprotein.
 3. The method of claim 1, wherein the antibody is a polyclonalantibody, and the antigen binding fragment is from a polyclonalantibody.
 4. The method of claim 1, wherein the antibody is a monoclonalantibody, and the antigen binding fragment is from a monoclonalantibody.
 5. The method of claim 1, wherein the antibody or antigenbinding fragment thereof is labeled with an agent.
 6. The method ofclaim 5, wherein the agent is selected from the group consisting of aradioisotope, fluorescent compound, bioluminescent compound,chemiluminescent compound, metal chelator, and enzyme.
 7. The method ofclaim 1, wherein the antigen binding fragment is an Fab, F(ab′)₂, Fv orsFv fragment.